Daphna Frenkiel-Krispin is a graduate student and Abraham Minsky is a group leader in the Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
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Biocrystallization: a Last-Resort Survival Strategy in Bacteria
With energy-dependent repair unavailable, special protein-DNA cocrystals enable prokaryotes to maintain genetic integrity and survive
Daphna Frenkiel-Krispin and Abraham Minsky
Efforts to curb and control bacterial infections are being recurrently frustrated by the emergence of pathogens that exhibit multiple drug resistance. The rapid spreading of resistant bacteria raises the specter of an imminent "postantimicrobials era," during which seemingly simple infections could turn into epidemics whose scope and severity may surpass those of microbial diseases in the preantibiotic days. These developments, exacerbated by a dearth of new classes of antibiotics and by the recent gruesome exploitation of bacterial pathogens for warfare purposes, underscore the urgent need for new insights into the factors that promote and sustain bacterial pathogenesis.
Pathogenesis is a continuous exercise in stress resistance exerted by both host and pathogens. For example, many intracellular pathogens, including Mycobacterium tuberculosis and virulent Salmonella species, are confined within host phagosomes in which the unicellular residents are subject to a wide assortment of harsh conditions and detrimental agents, including low oxygen levels, highly reactive oxygen and nitrogen intermediates, acidic pH, antimicrobial peptides, and nutritional deprivation. Similarly, extracellular pathogens such as Escherichia coli and Yersinia species regularly encounter extreme pH values, reactive electrophilic agents, and osmotic stress. In the ex vivo aquatic or soil habitats where bacteria persist as free-living organisms and disseminate, pathogens often are exposed to oxidative and osmotic stresses, potentially lethal doses of ultraviolet (UV) irradiation, high salinity, sharp temperature alterations, and deprivation of practically all nutrients. Thus, whether within or outside living hosts, bacteria continuously cope with rapidly altering, frequently hostile environments.
A cell's genetic complement is a primary target of environmental assaults. DNA molecules are intrinsically susceptible to damage by numerous physical and chemical agents, and even low-frequency DNA lesions can be lethal if unrepaired. These observations highlight the need for efficient DNA protection mechanisms. Eukaryotic systems preserve their DNA molecules through two distinct pathways: (i) enzymes, including catalases, superoxide dismutase, and glutathione peroxidase, neutralize detrimental chemicals, and (ii) a wide array of enzymes replaces altered nucleotides and restores the integrity of the genetic complement. The tight nucleosomal assembly, which physically protects DNA, supplements those two biochemical pathways.
This ever-present structural protection is, however, absent in prokaryotes, which lack a nucleosomal organization and whose chromatin is characterized by a low ratio of nonspecific DNA-binding proteins to DNA. All known strategies deployed for protecting DNA in nonsporulating prokaryotes rely on inducible enzymatic processes that are activated by specific signals. Since inducible responses need to cope with assaults that are already present, they must reach full capacity as fast as possible. Thus, bacterial stress responses typically are based on elaborate integrated circuits and branched regulatory cascades that enable a burst-like induction of one or several regulons in response to a particular assault. Activation of inducible stress responses is thus associated with rapid metabolic readjustments that require de novo synthesis of numerous enzymes, some in very large quantities.
Moreover, the actual repair functions of many of these enzymes are associated with massive energy consumption. Specifically, the exquisitely regulated activities of enzymes that participate in the SOS response to DNA breaks, as well as the activities of those involved in excision DNA repair pathways, are heavily ATP dependent, making such DNA repair activities fundamentally incompatible with states during which intracellular pools of high-energy compounds are severely depleted. Two such states are considered here: extensive starvation upon which energy-generating processes are impaired, and prolonged exposure to DNA-damaging agents that deplete energy because of the prevalence of massive repairs.
Bacteria experiencing these states display a novel mode of stress resistance: they form highly ordered, energy-independent intracellular assemblies within which DNA molecules are sequestered and structurally protected.
DNA Protection in Starved Bacteria
Microbial pathogens are starved regularly, making this stress a central facet of pathogenesis. Typically, such pathogens remain viable for long periods under conditions that are not propitious for their growth. Significantly, when starved, bacteria become resistant to harsh and diverse environmental assaults that would be lethal under normal growth conditions. It is generally believed that a small group of proteins mediates this enhanced endurance, or "maintenance mode." Typically, their synthesis begins at the onset of stationary phase and proceeds for several hours.
For the maintenance mode to be effective, these enzymes need to remain functional throughout starvation, and they also need a continuous supply of metabolic energy for the biochemical processes that they catalyze. Neither of these requirements can be effectively fulfilled during prolonged starvation. A growing body of evidence points to an accelerated oxidation and degradation of proteins during stationary phase. In particular, proteins that are directly involved in bacterial stress responses, including the heat shock protein DnaK and the DNA-binding protein H-NS, are especially susceptible to oxidative damage.
Meeting energy supply needs may be even more problematic during periods of prolonged starvation. For example, extensively starved bacteria degrade endogenous components such as membranes and ribosomes to provide a source of high-energy compounds. Thus, unless tightly regulated, enzymatic repair activities may, by themselves, lead to an irreversible loss of cell integrity or of essential cellular functions. Hence, inducible, chemically dependent DNA protection strategies that are effective during active growth become progressively ineffective upon severe depletion of nutrients.
Intracellular DNA-Dps Crystalline Assemblies Protect DNA
Under conditions of nutritional stress, E. coli produces a nonspecific DNA-binding protein termed Dps, whose expression is regulated by the alternative sigma factor RpoS. Dps accumulates to a very large amount, constituting the major component of the chromatin in late stationary bacteria. Within DNA-Dps complexes, the stability of Dps is dramatically enhanced relative to the stability of the free protein, and DNA molecules are effectively protected against oxidating agents and nucleases.
The avid binding between the Dps and DNA and the resulting enhanced protection of both components is notable, since Dps does not share sequence homology to any currently known DNA-binding protein and does not contain any recognizable DNA-binding motifs. The finding that the surface of the Dps dodecameric assembly, which is the putative DNA-binding species, is dominated by negative charges is intriguing in light of the negatively charged DNA backbone of nucleic acids. Close Dps homologs are found among many distantly related bacteria, suggesting that this protein maintains a general and crucial function.
The unusual features associated with Dps and its complex with nucleic acids, as well as its apparent abundance among diverse prokaryotes, prompted us to investigate whether novel mechanisms account for Dps-dependent DNA protection in starved bacteria. Purified Dps and DNA molecules interact very rapidly to form DNA-Dps cocrystals, within which DNA is effectively protected against oxidative agents and various nucleases (Fig. 1a). The rapid DNA-Dps cocrystallization occurs irrespective of DNA length (from 20 to 3,000 base pairs) or of DNA topology (supercoiled or linear). These complexes are distinct from those formed between DNA and nonspecific DNA-binding proteins such as RecA, sperm protamines, and histone H1, that tend to coat DNA molecules or form amorphous aggregates with it.
Thus, the rapidly formed DNA-Dps cocrystals represent an exceptional binding and protection mode. Is this complex physiologically relevant? Recent evidence suggests that it is.
For example, crystalline assemblies whose morphology is similar to in vitro DNA-Dps cocrystals can be detected in electron micrographs of starved E. coli mutant cells that slightly overproduce the Dps protein (Fig. 1b-d). In micrographs of starved wild-type bacteria we observe a dense mass in the center of their cytoplasm, from which the ribosomes are completely excluded.
To resolve the structure of this dense region, actively growing and starved intact E. coli cells were probed by X-ray scattering (Fig. 2a). The starved wild-type bacteria exhibit two X-ray bands. A relatively narrow band corresponds to a spacing of 94Å, and a broader band with two maxima, at 49Å and 47A. Actively growing cells did not exhibit any X-ray diffraction maxima. The diffraction patterns are consistent with a DNA-Dps structure in which Dps and DNA form stacked alternating layers (Fig. 2b). According to this model, the diffraction at 94A corresponds to the intralayer spacing between Dps dodecamers whose diameter is about 90A. The broad band centered at 47Å to 49Å likely represents a superposition of a second order Dps-Dps diffraction and DNA-DNA spacing.
On the basis of these X-ray scattering patterns and on Dps being the most abundant DNA-binding protein in starved E. coli cells, we concluded that the central region in starved wild-type bacteria consists of tightly packed DNA-Dps micro cocrystals. Within this tightly packed assembly, DNA molecules are structurally sequestered and physically protected.
These findings raise two interrelated questions: What is the nature of the signal that specifically induces ordered DNA-Dps complexes to form in starved bacteria? And, knowing that the surface of the Dps dodecamer is predominantly negatively charged, what is the actual mode of the DNA-Dps interaction? In vitro and in vivo studies indicate that the interaction between the two negatively charged species is mediated through multiple ion bridges that are maintained by doubly charged cations such as Mg2+ and Fe2+. Such ion bridges can be formed only within a particular concentration range of the doubly charged ions. Above or below this range, both DNA and Dps dodecamers surfaces are predominantly positively or negatively charged, respectively, and hence electrostatically repel each other.
Thus, as long as the growth medium contains nutrients that sustain fast and active growth, including divalent ions in relatively high concentrations, electrostatic repulsion prevents DNA-Dps complexes from forming. Yet, bacterial proliferation in defined niches within a host or in ex vivo environments progressively depletes divalent ions due to their rapid consumption or to host immune defense activities. As ion levels fall below a threshold value, ion bridges between DNA and Dps dodecamers can be formed, resulting in binding. When fresh nutrients—which include divalent ions—become available, DNA is released from the DNA-Dps cocrystals.
The proposed role of divalent ions as an on-off switch for DNA-Dps binding and cocrystallization is of fundamental physiological significance: upon bacterial infection, the host actively reduces the availability of iron ions in the phagosomes as part of its defence strategy against pathogens. Moreover, as phagosomes mature, Mg2+ ion levels decrease to very low levels within the phagosomal milieu. As such, DNA protection that is mediated through structural sequestration within DNA-Dps cocrystals, provides a new aspect to the notion that pathogenic bacteria have evolved effective means to exploit the host defense mechanisms.
Biocrystallization as a Response to DNA Damage
When bacteria are confronted with agents that cause double-stranded DNA breaks, cells mount an elaborate response. RecA, a DNA-binding protein whose cellular concentration substantially increases following DNA damage, plays a central role in this response.
For example, RecA is involved in the multistep DNA recombinational repair pathway. Initially, it coats a single-stranded DNA substrate, forming a presynaptic filament that acts as a sequence-specific DNA-binding entity. This filament is capable of searching and binding double-stranded DNA sites that are homologous to the RecA-coated segment. Within the resulting joint species, DNA strand exchange and heteroduplex extension processes are promoted. The mechanism that enables rapid searching for DNA homologies within such a crowded and complex genome remains enigmatic.
To reach its target, any sequence-specific DNA-binding protein must overcome two general obstacles: the minute cellular concentration of the target, and a vast excess of nontarget—yet still competitive—DNA sites. The search for a homologous DNA site conducted by the RecA-DNA presynaptic filament shares these hurdles, but is further encumbered by the uniquely adverse diffusion characteristics of its components. A DNA target corresponds to a segment that is part of, and embedded within, the chromosome. This and the large structural asymmetry of DNA minimizes the diffusion constant of DNA sites. In a homology search executed by the RecA-DNA filament, both the searching and the target entities are chromosomal DNA sites whose small diffusion constants drastically attenuate their encounter rate. How then does a RecA-mediated intracellular search evade the kinetic impediments that are intrinsic to the nature of its components? Moreover, since recombinational DNA repair processes are heavily ATP-dependent, prolonged exposure of bacteria to DNA-damaging agents is likely to result in a progressive depletion of the cellular ATP pools, and hence to render DNA repair increasingly inefficient. Are the cells doomed at this point?
Lateral DNA-RecA Coaggregates: a Potential Repairosome and Means for Protecting DNA
To address the issues of DNA homology search and DNA protection, we conducted electron microscopy studies on bacteria exposed to DNA-damaging factors such as UV-irradiation and various drugs. These studies revealed that E. coli cells exposed to assaults that lead to double-strand DNA breaks form a laterally ordered macroscopic assembly, which is detected several minutes after exposure (Fig. 3a). The aggregation is completely reversible, disappearing shortly after the damaging factors have been removed. Immunolabeling and specific DNA staining indicate that the intracellular aggregates consist of DNA and RecA molecules.
When measured in vitro, RecA-mediated homologous pairing exhibits Michaelis-Menten type kinetics, for which the rate-limiting step is conversion to products of a complex composed of RecA-DNA presynaptic filaments and double-stranded DNA molecules. Large RecA-DNA coaggregates can be detected by sedimentation and in vitro electron microscopy, and may be an intermediate on the pathway to homologous alignment. Presumably, transient protein-protein, protein-DNA, and DNA-DNA interactions hold such coaggregates together—in effect, reducing the sampling volume.
We propose that the intracellular assemblies represent a functional analog of the in vitro RecA-DNA networks. Being completely disordered, the in vitro networks cannot, however, alter the dimensionality of the search. In contrast, the intracellular assemblies may promote a homology search by both an attenuated sampling volume that results from RecA-DNA coaggregation, and a reduced dimensionality which specifically stems from the longitudinal organization of the in vivo assembly. Within such a linear, yet loosely packed and fluid lateral array consisting of chromosomal RecA-DNA filaments and double-stranded DNA, geometrical constraints facilitate translational movement. Thus, instead of sampling the genome through a three-dimensional random drift, RecA-DNA presynaptic filaments search for homologous sites by sliding along the double-stranded DNA, whose parallel arrangement coincides with the main axis of the assembly. We suggest calling this coaggregate a repairosome, indicating the actual intracellular site where RecA repairs damaged DNA.
When bacteria are exposed continuously to DNA-damaging agents, the loosely packed wavy assemblies that are detected following UV pulses or short exposure periods to nalidixic acid progressively assume a tight morphology. Diffraction patterns indicate the crystalline nature of these assemblies. Presumably, the accumulation of stable joints between presynaptic filaments and homologous double-stranded DNA segments that occurs upon continuous exposure results in an attenuated fluidity of the assemblies, thus enhancing their compactness and order. The additional density detected in the in vivo projection map in comparison to the map of the in vitro RecA crystal is accounted for by the presence of DNA in the intracellular crystals (Figure 3b-f).
The crystalline morphology that characterizes RecA-DNA assemblies in bacteria that have sustained prolonged exposure to DNA-damaging agents is, however, intriguing: such a tight organization cannot be reconciled with dynamic search and repair processes. It may be that the intracellular assemblies progressively assume a protective role, physically shielding DNA molecules. A progression of a structure-function correlation is thus suggested. A dynamic RecA-DNA assembly whose loose longitudinal organization allows for translational motion and hence for dynamic repair processes is initially formed. This repairosome is progressively transformed into a "real" RecA-DNA cocrystal, in which DNA molecules are structurally sequestered and protected.
Stress Resistance and Biocrystallization
Studying the mechanisms underlying the ability of bacteria to endure prolonged stress during periods when intracellular energy resources are severely depleted is a central theme to our research. Conventional bacterial stress responses depend on energy-consuming enzymatic transactions. Our observations imply that when cellular energy pools are depleted, a novel DNA protection strategy is deployed. When dynamic, energy-dependent, repair processes can no longer be effectively induced, survival derives from the ability of prokaryotes to maintain the integrity of their genetic complement through biocrystallization. This "last-resort" strategy consists of the formation of tightly packed and highly ordered structures, within which DNA molecules are, at least partially and temporally, protected through structural sequestration. The strategy entails the use of two stress-related bacterial crystalline assemblies: DNA-Dps cocrystals in response to starvation, and DNA-RecA cocrystals in response to extensive exposure to DNA-damaging agents.
In both cases, either because of prolonged starvation or during large-scale DNA repair processes in the aftermath of extensive UV-induced damage, cellular energy pools are depleted. Moreover, this type of biocrystallization is reversible. Both DNA-Dps and DNA-RecA cocrystals form in response to environmental stress, and both swiftly dissipate when that stress is alleviated and cells are no longer endangered.
Because these biocrystals form and persist without consuming metabolic energy, known antibiotics that interfere with metabolic enzymes are unlikely to perturb this process. Therefore, developing a deeper understanding of the factors that promote, prevent, or otherwise affect biocrystallization might lead to the design of novel antimicrobial agents.
ACKNOWLEDGMENTS
Our studies were supported by the Israel Science Foundation founded by the Academy of Sciences and Humanities, and by the Minerva Foundation, Germany.
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