Necrosis Is an Active and Controlled Form of Programmed Cell Death

In all studies on programmed cell death (PCD) and apoptosis as its most showy form, this process was considered to be a paradigmatic antithesis to necrotic cell death. On one hand, a concept on necrosis as a cellular cataclysm, an uncontrolled and passive phenomenon, had been provoked by an enormous bulk of experimental data on its inducibility by super-physiological exposures. On the other hand, much attention was attracted to a rapidly expanding (from nematodes) field of genetic studies on PCD. However, the findings accumulated which suggested a likeness rather than the opposition of the necrotic and apoptotic forms of elimination of unwanted cells. 1. Very diverse pathophysiological exposures (stimuli, stresses), such as heat, ionizing radiation, pathogens, cytokines cause both forms of cell death in the same cell population. 2. Antiapoptotic mechanisms (e.g., Bcl-2) can protect cells from both necrotic and apoptotic destruction. 3. Biochemical interventions (e.g., with inhibitors of poly-(ADP-riboso)-polymerase) into the signal and executive mechanisms of PCD can change the choice of the cell death form. 4. During both necrosis and epigenetic programs of apoptotic cell death that need no macromolecular synthesis (e.g., the CD95-dependent death), the nucleus plays a passive role. Therefore, necrosis, similarly to apoptosis, is suggested to be a form of the programmed cell death. However, for the whole body the physiological consequences of apoptosis and necrosis are quite different. In the case of apoptosis, all constituents of the nucleus and cytoplasm are isolated by an undamaged membrane and then by phagocytes together with the membrane-bound eat me markers (phosphatidylserine, etc.). In other words, the elimination of the cell which has realized its apoptotic program remains virtually unnoticed by the body. In the case of necrosis, the cytoplasmic content released into the intercellular space provokes an inflammatory response, i.e., an activation of resident phagocytes and attraction of leukocytes into the necrosis zone. It is suggested that under pathophysiological conditions, the necrotic cell destruction should amplify and catalyze pathological processes. The experimental data available now suggest that a disturbance in the body of optimal balance between the necrotic and apoptotic forms of PCD should be a crucial factor in the development of various pathophysiological processes associated with inflammation (diabetes, arthritis) or with aging (atherosclerosis, neurodegenerative diseases).     

A Conserved DNA Damage Response Pathway Responsible for Coupling the Cell Division Cycle to the Circadian and Metabolic Cycles

The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a “zeitgeber”, or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.     

mraY Is an Essential Gene for Cell Growth in Escherichia coli

The synthesis of the murein precursor lipid I is performed by MraY. We have shown that mraY is an essential gene for cell growth. Cells depleted of MraY first swell and then lyse. The expression of mraY DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K⁺, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K⁺ deprivation. Wild-type granule cells in 5 mM K⁺ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.     

The Escherichia coli mrsC Gene Is Required for Cell Growth and mRNA Decay

We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis. Originally designated mrsC (mRNA stability), the mrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F. Wang et al., J. Bacteriol. 180:1929–1938, 1998). Strains carrying the thermosensitive mrsC505 allele stopped growing soon after the temperature was shifted to 44°C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44°C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant. In an rne-1 mrsC505 double mutant, the average half-life was 19.8 min. Inactivating mrsC significantly increased the half-lives of the trxA, cat, secG, and kan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of mrsC505 multiple mutants at 44°C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.     

Mitochondrial Fragmentation Is Involved in Methamphetamine-Induced Cell Death in Rat Hippocampal Neural Progenitor Cells

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca²⁺) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.     

Cyclic AMP Phosphodiesterase in Leydig Cell Preparations of the Rat Testis

Enzymatic digestion of the interstitial tissue of early juvenile and adult rat testes resulted in an enrichment of the Leydig cell population. The cells of the intertubular preparation from adult testes were separated by centrifugal elutriation, according to differences in sedimentation velocity, a counter-flow centrifugation technique leading to 70% Leydig cell purity. Using this approach, it was possible to demonstrate that Leydig cells from adult testes contain only low affinity isoenzymes of cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17), an intracellular regulator of cAMP. Starch gel electrophoresis showed that the isozyme of cAMP PDE of Leydig cells is masked in crude testis homogenates due to the relatively low level of these cells in the total population. In Leydig cells, there are two different electrophoretic forms expressed which resemble two of eleven different molecular forms of cAMP PDE demonstrated for comparison in 21 different organs of the adult rat.
An interstitial cell preparation from early juvenile testes, with a Leydig cell content of up to 20%, was also investigated electrophoretically with regard to molecular forms of cAMP PDE, the properties of which were characterized by kinetic analysis of cAMP hydrolysis. The results presented are discussed in relation to the onset of testosterone synthesis in Leydig cells of prepubertal rats leading to the initiation of male puberty.     

Nucleotide Excision Repair Is a Predominant Mechanism for Processing Nitrofurazone-Induced DNA Damage in Escherichia coli

Nitrofurazone is reduced by cellular nitroreductases to form N²-deoxyguanine (N²-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N²-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.Replication in the presence of DNA damage is thought to produce most of the mutagenesis, genomic rearrangements, and lethality that occur in all cells. UV-induced photoproducts, X-ray-induced strand breaks, psoralen- or cis-platin-interstrand cross-links, oxidized bases from reactive oxygen species, and base depurination are just a few of the structurally distinct challenges that the replication machinery must overcome. It seems likely that the mechanisms that process these lesions will vary depending on the nature of the impediment.While a number of the lesions described above are known to block replication, the events associated with UV-induced damage have been the most extensively characterized. UV irradiation causes the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts in DNA that block the progression of the replication fork (16, 29, 30, 37). Following the arrest of replication at UV-induced damage, RecA and several RecF pathway proteins are required to process the replication fork such that the blocking lesion is removed or bypassed (2, 5, 6, 8-10). Cells lacking either RecA or any of several RecF pathway proteins are hypersensitive to UV-induced damage and fail to recover replication following disruption by the lesions (2, 6, 10). RecBCD is an exonuclease/helicase complex that is involved in repairing double-strand breaks (38). It also is required for resistance to UV-induced damage, although it is not required to process or restore disrupted replication forks, and the substrates it acts upon after UV irradiation currently remain unclear (3, 10, 19).Survival and the ability to resume DNA synthesis following UV-induced damage depend predominantly on the removal of the lesions by nucleotide excision repair (5, 7, 36). Cells deficient in nucleotide excision repair are unable to remove UV-induced DNA lesions and exhibit elevated levels of mutagenesis, strand exchanges, rearrangements, and cell lethality (16, 33, 34). In cases where replication fork processing or lesion repair is prevented, the recovery of replication and survival become entirely dependent on translesion synthesis by DNA polymerase V (Pol V) (6). However, in repair-proficient cells, the contribution of translesion synthesis to recovery and survival is minor and is detected only following UV doses that exceed the repair capacity of the cell (5, 6).Less is known about how replication recovers from other forms of DNA damage. We chose to characterize nitrofurazone, because a number of studies suggested that N²-deoxyguanine (N²-dG) adducts induced by this and other agents would be processed differently than UV-induced lesions. Nitrofurazone is a topical antibacterial agent that historically has been used for treating burns and skin grafts in patients and animals (14, 15, 32). Nitrofurazone toxicity is known to require activation by cellular nitroreductases (25, 42). However, the mechanism and targets of its antimicrobial properties have yet to be fully elucidated. In addition to its antimicrobial properties, the reduced nitrofurazone metabolites also target DNA and have been shown to induce free radical damage, strand breaks, and N²-dG adducts (26, 40, 42, 45), and they are mutagenic and carcinogenic in rodent models (1, 15, 24, 39).Whereas nucleotide excision repair is the predominant mechanism required for survival after UV-induced damage, a number of studies suggest that translesion synthesis plays a larger role in survival after nitrofurazone-induced DNA damage. dinB mutants lacking Pol IV were shown to be hypersensitive to nitrofurazone compared to cells that constitutively express the polymerase (17). Biochemically, Pol IV and a number of Pol IV homologs from other organisms have been shown to efficiently replicate over a range of N²-dG adducts in vitro (17, 35, 44). In addition, several studies have reported that uvrA mutants, which are defective in nucleotide excision repair, do not exhibit any hypersensitivity to nitrofurazone or other agents that induce similar adducts in vivo (12, 21, 27). Early studies also observed a direct correlation between nitrofurazone-induced mutations and lethality, suggesting that mutagenic lesions persist in the DNA to cause toxicity (21, 23, 27, 43). Consistent with these observations, nitrofuran-induced lesions were found to be poor substrates for nucleotide excision repair in vitro (46).Taken together, these observations suggest to us that the cellular response to nitrofurazone will be distinct from its response to UV irradiation. However, no study has examined the relative contributions that nucleotide excision repair, translesion synthesis, or recombination has in recovering from nitrofurazone-induced damage. In this study, we characterized the mechanism by which nitrofurazone inhibits DNA replication and identified the genes that contribute to the recovery, survival, and mutagenesis of Escherichia coli treated with nitrofurazone. In contrast to previous studies, we found that survival following nitrofurazone-induced damage depends predominantly on nucleotide excision repair. Similarly to UV-induced DNA damage, both the RecF and RecBC pathways contribute to survival following nitrofurazone-induced DNA damage. The contribution of translesion polymerases to survival was minor and was mediated by Pol IV. In addition, we found that nitrofurazone can act to inhibit DNA replication directly when used at higher concentrations. The direct inhibition of replication is reversible and occurs independently of DNA damage, suggesting that DNA is not the primary target of its antimicrobial properties.     

Toxin-Antitoxin Systems Are Important for Niche-Specific Colonization and Stress Resistance of Uropathogenic Escherichia coli

Toxin-antitoxin (TA) systems are prevalent in many bacterial genomes and have been implicated in biofilm and persister cell formation, but the contribution of individual chromosomally encoded TA systems during bacterial pathogenesis is not well understood. Of the known TA systems encoded by Escherichia coli, only a subset is associated with strains of extraintestinal pathogenic E. coli (ExPEC). These pathogens colonize diverse niches and are a major cause of sepsis, meningitis, and urinary tract infections. Using a murine infection model, we show that two TA systems (YefM-YoeB and YbaJ-Hha) independently promote colonization of the bladder by the reference uropathogenic ExPEC isolate CFT073, while a third TA system comprised of the toxin PasT and the antitoxin PasI is critical to ExPEC survival within the kidneys. The PasTI TA system also enhances ExPEC persister cell formation in the presence of antibiotics and markedly increases pathogen resistance to nutrient limitation as well as oxidative and nitrosative stresses. On its own, low-level expression of PasT protects ExPEC from these stresses, whereas overexpression of PasT is toxic and causes bacterial stasis. PasT-induced stasis can be rescued by overexpression of PasI, indicating that PasTI is a bona fide TA system. By mutagenesis, we find that the stress resistance and toxic effects of PasT can be uncoupled and mapped to distinct domains. Toxicity was specifically linked to sequences within the N-terminus of PasT, a region that also promotes the development of persister cells. These results indicate discrete, multipurpose functions for a TA-associated toxin and demonstrate that individual TA systems can provide bacteria with pronounced fitness advantages dependent on toxin expression levels and the specific environmental niche occupied.     

Properties of a Cell Fraction That Repairs Damage to the Cell Division Mechanism of Escherichia coli

A factor in bacterial cell extracts which induces cell division in filaments of Escherichia coli produced by irradiation was found to be associated with a heat-labile particulate fraction which sediments at about 100S. Frozen or lyophilized samples of the cell extracts were stable for considerable periods of time. Fractions purified by centrifugation contained 2% of the total protein of the extract but no measurable ribonucleic acid or deoxyribonucleic acid. The extracts were inactivated by incubation with phospholipase A, lipases, and detergents, but not by incubation with selected nucleases and proteases.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.     

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.     

Castration Increases Cell Damage Induced by Porphyrins in the Harderian Gland of Male Syrian Hamster. Necrosis and Not Apoptosis Mediates the Subsequent Cell Death

It is known that the Harderian gland of male Syrian hamster synthesizes a much smaller amount of porphyrins than the gland of the female and that castration greatly increases this synthesis. We have studied in this experimental model the behavior of the different classes of secretory cells and their role in the synthesis of porphyrins, attempting to clarify the participation of these compounds in the cell damage leading to the formation of clear cells previously described in the gland of females. We have also investigated the mechanism underlying the death of these secretory cells after porphyrin accumulation (necrosis vs apoptosis). To achieve this, we have utilized the following techniques: (a) morphometrical; (b) ultrastructural; (c) biochemical (fluorescence spectrophotometry); and (d) molecular (DNA nick-end labeling in methacrylate sections and dot blot analysis). The glands from male hamsters (serving as control) present a very low rate of damaged cells that progressively rises after castration. This rise runs parallel to that of porphyrin synthesis, porphyrin deposits, and the decrease of Type II secretory cells. The damage and subsequent death of the secretory cells in the gland is produced by the deposit of porphyrins in the mitochondrial membrane. This porphyrin accumulation leads to a complete mitochondrial destruction that finally results in cell death and its secretion into the lumen. We finally conclude that this event is not a physiological cell death (apoptosis) but the consequence of the toxic accumulation of porphyrins (necrosis).     

Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from Escherichia coli Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine

The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.