Steven E. Finkel

Long-term survival during stationary phase: evolution and the GASP phenotype

The traditional view of the stationary phase of the bacterial life cycle, obtained using standard laboratory culture practices, although useful, might not always provide us with the complete picture. Here, the traditional three phases of the bacterial life cycle are expanded to include two additional phases: death phase and long-term stationary phase. In many natural environments, bacteria probably exist in conditions more akin to those of long-term stationary-phase cultures, in which the expression of a wide variety of stress-response genes and alternative metabolic pathways is essential for survival. Furthermore, stressful environments can result in selection for mutants that express the growth advantage in stationary phase (GASP) phenotype.

DNA protection by stress-induced biocrystallization

The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps,. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA–Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.

Dps Protects Cells against Multiple Stresses during Stationary Phase

Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli. Dps homologs are found throughout the bacteria and in at least one archaeal species. Dps has been shown to protect cells from oxidative stress during exponential-phase growth. During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal. We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells. We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock. The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression.

DNA as a Nutrient: Novel Role for Bacterial Competence Gene Homologs

The uptake and stable maintenance of extracellular DNA, genetic transformation, is universally recognized as a major force in microbial evolution. We show here that extracellular DNA, both homospecific and heterospecific, can also serve as the sole source of carbon and energy supporting microbial growth. Mutants unable to consume DNA suffer a significant loss of fitness during stationary-phase competition. In Escherichia coli, the use of DNA as a nutrient depends on homologs of proteins involved in natural genetic competence and transformation in Haemophilus influenzae and Neisseria gonorrhoeae. Homologs of these E. coli genes are present in many members of the gamma subclass of Proteobacteria, suggesting that the mechanisms for consumption of DNA may have been widely conserved during evolution.

SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness

Escherichia coli encodes three SOS-induced DNA polymerases: pol II, pol IV, and pol V. We show here that each of these polymerases confers a competitive fitness advantage during the stationary phase of the bacterial life cycle, in the absence of external DNA-damaging agents known to induce the SOS response. When grown individually, wild-type and SOS pol mutants exhibit indistinguishable temporal growth and death patterns. In contrast, when grown in competition with wild-type E. coli, mutants lacking one or more SOS polymerase suffer a severe reduction in fitness. These mutants also fail to express the “growth advantage in stationary phase” phenotype as do wild-type strains, instead expressing two additional new types of “growth advantage in stationary phase” phenotype. These polymerases contribute to survival by providing essential functions to ensure replication of the chromosome and by generating genetic diversity.

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.

The Growth Advantage in Stationary-Phase Phenotype Conferred by rpoS Mutations Is Dependent on the pH and Nutrient Environment

Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent.

Simultaneous analysis of physiological and electrical output changes in an operating microbial fuel cell with Shewanella oneidensis.

Changes in metabolism and cellular physiology of facultative anaerobes during oxygen exposure can be substantial, but little is known about how these changes connect with electrical current output from an operating microbial fuel cell (MFC). A high‐throughput voltage based screening assay (VBSA) was used to correlate current output from a MFC containing Shewanella oneidensis MR‐1 to carbon source (glucose or lactate) utilization, culture conditions, and biofilm coverage over 250 h. Lactate induced an immediate current response from S. oneidensis MR‐1, with both air‐exposed and anaerobic anodes throughout the duration of the experiments. Glucose was initially utilized for current output by MR‐1 when cultured and maintained in the presence of air. However, after repeated additions of glucose, the current output from the MFC decreased substantially while viable planktonic cell counts and biofilm coverage remained constant suggesting that extracellular electron transfer pathways were being inhibited. Shewanella maintained under an anaerobic atmosphere did not utilize glucose consistent with literature precedents. Operation of the VBSA permitted data collection from nine simultaneous S. oneidensis MR‐1 MFC experiments in which each experiment was able to demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a carbon electrode. These data provide the first direct evidence of complex cellular responses to electron donor and oxygen tension by Shewanella in an operating MFC at select time points. Biotechnol. Bioeng. 2009;103: 524–531. Published 2009 Wiley Periodicals, Inc.

Characterization of electrochemically active bacteria utilizing a high‐throughput voltage‐based screening assay

Metal reduction assays are traditionally used to select and characterize electrochemically active bacteria (EAB) for use in microbial fuel cells (MFCs). However, correlating the ability of a microbe to generate current from an MFC to the reduction of metal oxides has not been definitively established in the literature. As these metal reduction assays may not be generally reliable, here we describe a four‐ to nine‐well prototype high throughput voltage‐based screening assay (VBSA) designed using MFC engineering principles and a universal cathode. Bacterial growth curves for Shewanella oneidensis strains DSP10 and MR‐1 were generated directly from changes in open circuit voltage and current with five percent deviation calculated between each well. These growth curves exhibited a strong correlation with literature doubling times for Shewanella indicating that the VBSA can be used to monitor distinct fundamental properties of EAB life cycles. In addition, eight different organic electron donors (acetate, lactate, citrate, fructose, glucose, sucrose, soluble starch, and agar) were tested with S. oneidensis MR‐1 in anode chambers exposed to air. Under oxygen exposure, we found that current was generated in direct response to additions of acetate, lactate, and glucose. Biotechnol. Bioeng. 2009;102: 436–444.

Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV.

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N2-N2-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5′ to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3′ to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N2-N2-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N2-N2-guanine ICLs.