Effects of prolonged exposure to moderate static magnetic field and its synergistic effects with alkaline pH on Enterococcus faecalis.

Static magnetic field (SMF) has been shown to biologically affect various microorganisms, but its effects on Enterococcus faecalis, which is associated with multiple dental infections, have not been reported yet. Besides, Enterococcus faecalis was found to be resistant to the alkaline environment provided by a major dental antimicrobial, calcium hydroxide. Therefore, the antibacterial activity of prolonged exposure to moderate SMF (170 mT) and its possible synergistic activity with alkaline pH (pH = 9) were evaluated in the study. The ability to form a biofilm under these conditions was examined by crystal violet assay. Real-time quantitative PCR was performed to evaluate the relative expression of stress (dnaK and groEL) and virulence (efaA, ace, gelE and fsrC) related genes. As the results indicated, cell proliferation was inhibited after 120 h of SMF exposure. What's more, the combined treatment of SMF and alkaline pH showed significantly improved antimicrobial action when compared to single SMF and alkaline pH treatment for more than 24 h and 72 h respectively. However, the ability to form a biofilm was also enhanced under SMF and alkaline pH treatments. SMF can induce stress response by up-regulating the expression of dnaK and elevate virulence gene expression (efaA and ace). These responses were more significant and more genes were up-regulated including groEL, gelE and fsrC when exposed to SMF and alkaline pH simultaneously. Hence, combination of SMF and alkaline pH could be a promising disinfection strategy in dental area and other areas associated with Enterococcus faecalis infections.

Antibacterial Mechanism of 405-Nanometer Light-Emitting Diode against Salmonella at Refrigeration Temperature.

The aim of this study was to elucidate the antibacterial mechanism of 405 ± 5-nm light-emitting diode (LED) illumination against Salmonella at 4°C in phosphate-buffered saline (PBS) by determining endogenous coproporphyrin content, DNA oxidation, damage to membrane function, and morphological change. Gene expression levels, including of oxyR, recA, rpoS, sodA, and soxR, were also examined to understand the response of Salmonella to LED illumination. The results showed that Salmonella strains responded differently to LED illumination, revealing that S. enterica serovar Enteritidis (ATCC 13076) and S. enterica subsp. enterica serovar Saintpaul (ATCC 9712) were more susceptible and resistant, respectively, than the 16 other strains tested. There was no difference in the amounts of endogenous coproporphyrin in the two strains. Compared with that in nonilluminated cells, the DNA oxidation levels in illuminated cells increased. In illuminated cells, we observed a loss of efflux pump activity, damage to the glucose uptake system, and changes in membrane potential and integrity. Transmission electron microscopy revealed a disorganization of chromosomes and ribosomes due to LED illumination. The levels of the five genes measured in the nonilluminated and illuminated S Saintpaul cells were upregulated in PBS at a set temperature of 4°C, indicating that increased gene expression levels might be due to a temperature shift and nutrient deficiency rather than to LED illumination. In contrast, only oxyR in S Enteritidis cells was upregulated. Thus, different sensitivities of the two strains to LED illumination were attributed to differences in gene regulation.IMPORTANCE Bacterial inactivation using visible light has recently received attention as a safe and environmentally friendly technology, in contrast with UV light, which has detrimental effects on human health and the environment. This study was designed to understand how 405 ± 5-nm light-emitting diode (LED) illumination kills Salmonella strains at refrigeration temperature. The data clearly demonstrated that the effectiveness of LED illumination on Salmonella strains depended highly on the serotype and strain. Our findings also revealed that its antibacterial mechanism was mainly attributed to DNA oxidation and a loss of membrane functions rather than membrane lipid peroxidation, which has been proposed by other researchers who studied the antibacterial effect of LED illumination by adding exogenous photosensitizers, such as chlorophyllin and hypericin. Therefore, this study suggests that the detailed antibacterial mechanisms of 405-nm LED illumination without additional photosensitizers may differ from that by exogenous photosensitizers. Furthermore, a change in stress-related gene regulation may alter the susceptibility of Salmonella cells to LED illumination at refrigeration temperature. Thus, our study provides new insights into the antibacterial mechanism of 405 ± 5-nm LED illumination on Salmonella cells.

Costs and benefits of natural transformation in Acinetobacter baylyi.

BACKGROUND: Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. RESULTS: We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. CONCLUSIONS: Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.

Response of genes related to iron and porphyrin transport in Porphyromonas gingivalis to blue light.

BACKGROUND: Antimicrobial blue light (aBL) kills a variety of bacteria, including Porphyromonas gingivalis. However, little is known about the transcriptomic response of P. gingivalis to aBL therapy. This study was designed to evaluate the selective cytotoxicity of aBL against P. gingivalis over human cells and to further investigate the genetic response of P. gingivalis to aBL at the transcriptome level. METHODS: Colony forming unit (CFU) testing, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to investigate the antimicrobial effectiveness of blue light against P. gingivalis. The temperatures of the irradiated targets were measured to prevent overheating. Multiple fluorescent probes were used to quantify reactive oxygen species (ROS) generation after blue-light irradiation. RNA sequencing (RNA-seq) was used to investigate the changes in global gene expression. Following the screening of target genes, real-time quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the regulation of gene expression. RESULTS: A 405 nm aBL at 100 mW/cm2 significantly killed P. gingivalis within 5 min while sparing human gingival fibroblasts (HGFs). No obvious temperature changes were detected in the irradiated surface under our experimental conditions. RNA-seq showed that the transcription of multiple genes was regulated, and RT-qPCR revealed that the expression levels of the genes RgpA and RgpB, which may promote heme uptake, as well as the genes Ftn and FetB, which are related to iron homeostasis, were significantly upregulated. The expression levels of the FeoB-2 and HmuR genes, which are related to hydroxyl radical scavenging, were significantly downregulated. CONCLUSIONS: aBL strengthens the heme uptake and iron export gene pathways while reducing the ROS scavenging pathways in P. gingivalis, thus improving the accumulation of endogenous photosensitizers and enhancing oxidative damage to P. gingivalis.

The Regulation of LexA on UV-Induced SOS Response in Myxococcus xanthus Based on Transcriptome Analysis.

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

Efficacy of UVC-LED in water disinfection on Bacillus species with consideration of antibiotic resistance issue.

Ultraviolet light emitting diode (UV-LED) has attracted extensive attention as a new technology to replace traditional mercury lamp for water disinfection. This study reported for the first time the application of UVC-LEDs in range of 200-280 nm for the treatment of two Gram-positive tetracycline resistant bacteria (TRB) from Bacillus species and their tetracycline resistant gene (TRG). The results showed that UVC-LEDs can inactivate TRB up to 5.7-log and inhibit TRG expression, especially at 268 nm. The required fluence was approximate to that of the referential non-resistant bacteria using the same UVC-LED, but far less than that of TRB using mercury lamp. After UVC-LED irradiation, photoreactivation was the dominant mechanism to repair TRB, just like non-resistant bacteria. But contrary to non-resistant bacteria, the regrowth ratio of TRB was remarkably high at 24 h since the end of the irradition, nevertheless the number of the regrown bacteria in the irradiated water was still less than that in the non-irradiated water. Whereas TRB restored resistance after repair even applying 268 nm at a fluence up to 46.08 mJ/cm2 (maximum in this study). This study highlights the merits of UVC-LED to effectively inactivate TRB in a prompt, energy-efficient and resistance-reducing way, while future study on TRB regrowth and resistance resilience is needed.

Microwave-induced release and degradation of airborne antibiotic resistance genes (ARGs) from Escherichia coli bioaerosol based on microwave absorbing material.

Antibiotic resistance genes (ARGs) have been detected in the atmosphere. Airborne ARGs transmission threatens human health. In the present study, we investigated the release and degradation of airborne ARGs from Escherichia coli bioaerosol through microwave (MW) irradiation. In this study, a new MW absorbing material (Fe3O4@SiC ceramic foam) that contributed to its stronger MW absorption is presented. When the MW input energy density was 7.4 × 103 kJ/m3, the concentration of airborne Escherichia coli decreased by 4.4 log. Different DNA forms were found in the air because MW irradiation ruptured cell membranes. The bound particles provide more protection for bound DNA in the degradation process than free DNA. After the self-degradation of the released airborne free ARGs, some of them would remain and continue to spread in the atmosphere. The released airborne free ARGs cannot be ignored. Total ARGs concentrations decrease rapidly with increased temperature. The inactivation rate constant of ARGs through MW irradiation is higher than that through the Fenton and UV, however, the energy efficiency per order of MW irradiation is lower. Therefore, MW irradiation with Fe3O4@SiC ceramic foam could efficiently degrade the distribution of ARGs in the atmosphere.

Genetic and physiological effects of noncoherent visible light combined with hydrogen peroxide on Streptococcus mutans in biofilm.

Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H(2)O(2)) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H(2)O(2) on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm(2)) in the presence of 3 to 300 mM H(2)O(2). The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H(2)O(2) on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H(2)O(2), has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H(2)O(2) treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H(2)O(2) to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.

The role of operating parameters and oxidative damage mechanisms of advanced chemical oxidation processes in the combat against antibiotic-resistant bacteria and resistance genes present in urban wastewater.

An upsurge in the study of antibiotic resistance in the environment has been observed in the last decade. Nowadays, it is becoming increasingly clear that urban wastewater is a key source of antibiotic resistance determinants, i.e. antibiotic-resistant bacteria and antibiotic resistance genes (ARB&ARGs). Urban wastewater reuse has arisen as an important component of water resources management in the European Union and worldwide to address prolonged water scarcity issues. Especially, biological wastewater treatment processes (i.e. conventional activated sludge), which are widely applied in urban wastewater treatment plants, have been shown to provide an ideal environment for the evolution and spread of antibiotic resistance. The ability of advanced chemical oxidation processes (AOPs), e.g. light-driven oxidation in the presence of H2O2, ozonation, homogeneous and heterogeneous photocatalysis, to inactivate ARB and remove ARGs in wastewater effluents has not been yet evaluated through a systematic and integrated approach. Consequently, this review seeks to provide an extensive and critical appraisal on the assessment of the efficiency of these processes in inactivating ARB and removing ARGs in wastewater effluents, based on recent available scientific literature. It tries to elucidate how the key operating conditions may affect the process efficiency, while pinpointing potential areas for further research and major knowledge gaps which need to be addressed. Also, this review aims at shedding light on the main oxidative damage pathways involved in the inactivation of ARB and removal of ARGs by these processes. In general, the lack and/or heterogeneity of the available scientific data, as well as the different methodological approaches applied in the various studies, make difficult the accurate evaluation of the efficiency of the processes applied. Besides the operating conditions, the variable behavior observed by the various examined genetic constituents of the microbial community, may be directed by the process distinct oxidative damage mechanisms in place during the application of each treatment technology. For example, it was shown in various studies that the majority of cellular damage by advanced chemical oxidation may be on cell wall and membrane structures of the targeted bacteria, leaving the internal components of the cells relatively intact/able to repair damage. As a result, further in-depth mechanistic studies are required, to establish the optimum operating conditions under which oxidative mechanisms target internal cell components such as genetic material and ribosomal structures more intensively, thus conferring permanent damage and/or death and preventing potential post-treatment re-growth.

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.

H2O2 and/or TiO2 photocatalysis under UV irradiation for the removal of antibiotic resistant bacteria and their antibiotic resistance genes.

Inactivating antibiotic resistant bacteria (ARB) and removing antibiotic resistance genes (ARGs) are very important to prevent their spread into the environment. Previous efforts have been taken to eliminate ARB and ARGs from aqueous solution and sludges, however, few satisfying results have been obtained. This study investigated whether photocatalysis by TiO2 was able to reduce the two ARGs, mecA and ampC, within the host ARB, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. The addition of H2O2 and matrix effect on the removal of ARB and ARGs were also studied. TiO2 thin films showed great effect on both ARB inactivation and ARGs removal. Approximately 4.5-5.0 and 5.5-5.8 log ARB reductions were achieved by TiO2 under 6 and 12mJ/cm2 UV254 fluence dose, respectively. For ARGs, 5.8 log mecA reduction and 4.7 log ampC reduction were achieved under 120mJ/cm2 UV254 fluence dose in the presence of TiO2. Increasing dosage of H2O2 enhanced the removal efficiencies of ARB and ARGs. The results also demonstrated that photocatalysis by TiO2 was capable of removing both intracellular and extracellular forms of ARGs. This study provided a potential alternative method for the removal of ARB and ARGs from aqueous solution.

Elimination of antibiotic-resistance bacterium and its associated/dissociative blaTEM-1 and aac(3)-II antibiotic-resistance genes in aqueous system via photoelectrocatalytic process.

The ubiquity of antibiotic-resistance bacteria (ARB) and antibiotic-resistance genes (ARGs) in various environmental matrices is a potential threat to human and ecological health. Therefore, the inactivation of ARB E. coli S1-23 and the elimination of its associated ARGs, blaTEM-1 and aac(3)-II, were investigated using the photoelectrocatalytic (PEC) process. Results indicate that the ARB E. coli S1-23 (1 × 108 cfu mL-1) and its ARGs (extracellular and intracellular) could be fully inactivated within 10 and 16 h PEC treatment, respectively. In contrast, photocatalytic (PC) and electrochemical (EC) treatments displayed no obvious effect; however, ARG-containing DNA extracted from E. coli S1-23, which was used as a model for dissociative naked ARGs, could be completely decomposed within a few minutes through these three treatments. Further analyses, including PCR, AFM and HPLC, proved that the structural integrity and surface topography of naked ARGs are damaged during treatment and can be completely eliminated. Furthermore, there is no generation of cytosine, guanine, adenine or thymine intermediates during the PEC, PC, and EC treatments. This study is the first report to propose the PEC treatment as a promising method for complete decomposition of ARB and ARGs in aqueous systems.

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain.

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen.

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.

Deletions induced by gamma rays in the genome of Escherichia coli.

An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ. This suggested that this origin of replication was involved in the process by which the deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system.

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.

Mutagenic specificity of ultraviolet light.

Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v. stimulates many types of mutations. In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5%. A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations. Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions. G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines. In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions. Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence. Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs. No additions were detected. The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1). In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence. For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots. The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion.

Intragenic domains of strand-specific repair in Escherichia coli.

Heterogeneity of DNA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and DNA strands. If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo. Here we measure the frequency of UV-induced cyclobutane pyrimidine dimers at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation. Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites. In lacZ, the domain of faster repair began 32 bases downstream of the transcription start site and required the mfd gene. Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo. Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (2) induction of lacZ transcription with isopropyl-beta-D-thiogalactoside overcame this reduction. Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.

Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity.

We showed that sufficient overexpression of the wild-type recF gene interfered with three normal cell functions: (1) UV induction of transcription from the LexA-protein-repressed sulA promoter, (2) UV resistance and (3) cell viability at 42 degrees. To show this, we altered a low-level overexpressing recF+ plasmid with a set of structurally neutral mutations that increased the rate of expression of recF. The resulting high-level overexpressing plasmid interfered with UV induction of the sulA promoter, as did the low-level overexpressing plasmid. It also reduced UV resistance more than its low level progenitor and decreased viability at 42 degrees, an effect not seen with the low-level plasmid. We used the high-level plasmid to test four recF structural mutations for residual activity. The structural alleles consisted of an insertion mutation, two single amino acid substitution mutations and a double amino acid substitution mutation. On the Escherichia coli chromosome the three substitution mutations acted similarly to a recF deletion in reducing UV resistance in a recB21 recC22 sbcB15 sbcC201 genetic background. By this test, therefore, all three appeared to be null alleles. Measurements of conjugational recombination revealed, however, that the three substitution mutations may have residual activity. On the high-level overexpressing plasmid all three substitution mutations definitely showed partial activity. By contrast, the insertion mutation on the high-level overexpressing plasmid showed no partial activity and can be considered a true null mutation. One of the substitutions, recF143, showed a property attributable to a leaky mutation. Another substitution, recF4101, may block selectively two of the three interference phenotypes, thus allowing us to infer a mechanism for them.

Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC.

First identified as an essential component of the phi X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec-, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.