umuC Gene Research Articles

Coexistence of plasmid-mediated tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in Klebsiella quasipneumoniae.

Klebsiella quasipneumoniae is a potential pathogen that has not been studied comprehensively. The emergence of multidrug-resistant (MDR) K. quasipneumoniae, specifically strains resistant to tigecycline and carbapenem, presents a significant challenge to clinical treatment. This investigation aimed to characterize MDR K. quasipneumoniae strain FK8966, co-carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 by plasmids. It was observed that FK8966's MDR was primarily because of the IncHI1B-like plasmid co-carrying tmexCD2-toprJ2 and blaIMP-4, and an IncFIB(K)/IncFII(K) plasmid harboring blaNDM-1. Furthermore, the phylogenetic analysis revealed that IncHI1B-like plasmids carrying tmexCD2-toprJ2 were disseminated among different bacteria, specifically in China. Additionally, according to the comparative genomic analysis, the MDR regions indicated that the tmexCD2-toprJ2 gene cluster was inserted into the umuC gene, while blaIMP-4 was present in transposon TnAs3 linked to the class 1 integron (IntI1). It was also observed that an ΔTn3000 insertion with blaNDM-1 made a novel blaNDM-1 harboring IncFIB(K)/IncFII(K) plasmid. The antimicrobial resistance prevalence and phylogenetic analyses of K. quasipneumoniae strains indicated that FK8966 is a distinct MDR branch of K. quasipneumoniae. Furthermore, CRISPR-Cas system analysis showed that many K. quasipneumoniae CRISPR-Cas systems lacked spacers matching the two aforementioned novel resistance plasmids, suggesting that these resistance plasmids have the potential to disseminate within K. quasipneumoniae. Therefore, the spread of MDR K. quasipneumoniae and plasmids warrants further attention.IMPORTANCEThe emergence of multidrug-resistant K. quasipneumoniae poses a great threat to clinical care, and the situation is exacerbated by the dissemination of tigecycline- and carbapenem-resistant genes. Therefore, monitoring these pathogens and their resistance plasmids is urgent and crucial. This study identified tigecycline- and carbapenem-resistant K. quasipneumoniae strain, FK8966. Furthermore, it is the first study to report the coexistence of tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in K. quasipneumoniae. Moreover, the CRISPR-Cas system of many K. quasipneumoniae lacks spacers that match the plasmids carried by FK8966, which are crucial for mediating resistance against tigecycline and carbapenems, indicating their potential to disseminate within K. quasipneumoniae.

Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

Persistent ciprofloxacin exposure induced the transformation of Klebsiella pneumoniae small colony variant into mucous phenotype

IntroductionSmall colony variant (SCV) is a bacterial phenotype closely related to persistent and recurrent infections. SCVs are mutations that occur within bacterial populations, resulting in a change in bacterial morphology and the formation of small colonies. This morphological change may enhance bacterial resistance to antibiotics and contribute to persistent and recurrent infections.MethodsWe isolated Klebsiella pneumoniae (KPN) and its SCV from a child with recurrent respiratory tract infections. KPN and SCV were treated with subinhibitory concentrations of antibiotics. growth curves, serum resistance experiments, macrophage phagocytosis experiments and whole genome sequencing were used to characterize KPN and SCV.ResultsAfter treating KPN and SCV with subinhibitory concentrations of antibiotics, we found that ciprofloxacin induced the SCV transition to the mucoid phenotype. We found that the growth of mucoid Klebsiella pneumoniae was significantly slower than maternal strain and SCV though growth curves. Serum resistance experiments showed that mucoid strains had significantly higher serum resistance compared to maternal strain and SCV. Macrophage phagocytosis experiments revealed that SCV had significantly higher intracellular survival rates compared to maternal strain and mucoid strains. Differential gene analysis of three strains revealed that the mucoid strain contained DNA polymerase V subunit UmuC gene on the plasmid, while the SCV strain had an additional IcmK family IV secretion protein on its plasmid.DiscussionOur study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. The higher resistance of serum of mucoid colonies was possibly related to the UmuC gene, while the increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

Integrative conjugative elements mediate the high prevalence of tmexCD3-toprJ1b in Proteus spp. of animal source.

Integrative conjugative elements (ICEs) are important mobile elements that are associated with the dissemination of antibiotic resistance genes (ARGs) in Proteus. Recent studies demonstrated that the tigecycline resistance gene cluster tmexCD-toprJ has emerged in ICEs of Proteus. However, the prevalence of tmexCD-toprJ positive Proteus from animal sources is unclear. To cover the gap, a total of 762 Proteus spp. were isolated from animal source from six provinces of China to identify the tmexCD-toprJ positive isolates. Eight tmexCD-toprJ positive isolates were identified, with the tmexCD-toprJ positive rate of 1.05% against all Proteus spp. and 2.79% against ICE-bearing Proteus spp. The tmexCD-toprJ gene cluster in these Proteus spp. were tmexCD3-toprJ1b and all of them were carried by ICEs. Genetic structure analysis showed that tmexCD3-toprJ1b-bearing ICEs were complicated and plastic, but the tmexCD3-toprJ1b was specifically integrated into variable region III (VRIII) of ICEs with the help of integrases. Furthermore, we found that the umuC gene, which is presented in VRIII of ICEs and other genetic structures of many other bacterial genomes, was a hotspot for the integration of tmexCD-toprJ-bearing potential mobile elements. In summary, our results suggested that Proteus were important reservoirs for tmexCD-toprJ due to the high prevalence of ICEs. Therefore, continuous surveillance of ICEs-associated ARGs in Proteus is necessary and significant for controlling their future transmission. IMPORTANCE The emergence and spread of tmexCD-toprJ have greatly weakened the function of tigecycline. Although studies have demonstrated the significance of Proteus as carriers for tmexCD-toprJ, the epidemic mechanism and characteristics of tmexCD-toprJ in Proteus remain unclear. Herein, we deciphered that the umuC gene in VRIII of SXT/R391 ICEs was a hotspot for the integration of tmexCD3-toprJ1b-bearing mobile genetic elements by genomic analysis. The mobilization and dissemination of tmexCD3-toprJ1b in Proteus were mediated by highly prevalent ICEs. Furthermore, the co-occurrence of tmexCD3-toprJ1b-bearing ICEs with other chromosomally encoded multidrug resistance gene islands warned that the chromosomes of Proteus are significant reservoirs of ARGs. Overall, our results provide significant insights for the prevention and control of tmexCD3-toprJ1b in Proteus.

Bioactivation mechanisms of N-hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids.

Aristolochic acids (AAs) are human nephrotoxins and carcinogens found in concoctions of Aristolochia plants used in traditional medicinal practices worldwide. Genotoxicity of AAs is associated with the formation of active species catalyzed by metabolic enzymes, the full repertoire of which is unknown. Recently, we provided evidence that sulfonation is important for bioactivation of AAs. Here, we employ Salmonella typhimurium umu tester strains expressing human N‐acetyltransferases (NATs) and sulfotransferases (SULTs), to study the role of conjugation reactions in the genotoxicities of N‐hydroxyaristolactams (AL‐I‐NOH and AL‐II‐NOH), metabolites of AA‐I and AA‐II. Both N‐hydroxyaristolactams show stronger genotoxic effects in umu strains expressing human NAT1 and NAT2, than in the parent strain. Additionally, AL‐I‐NOH displays increased genotoxicity in strains expressing human SULT1A1 and SULT1A2, whereas AL‐II‐NOH shows enhanced genotoxicity in SULT1A1/2 and SULT1A3 strains. 2,6‐Dichloro‐4‐nitrophenol, SULTs inhibitor, reduced umuC gene expression induced by N‐hydroxyaristolactams in SULT1A2 strain. N‐hydroxyaristolactams are also mutagenic in parent strains, suggesting that an additional mechanism(s) may contribute to their genotoxicities. Accordingly, using putative SULT substrates and inhibitors, we found that cytosols obtained from human kidney HK‐2 cells activate N‐hydroxyaristolactams in aristolactam‐DNA adducts with the limited involvement of SULTs. Removal of low‐molecular‐weight reactants in the 3.5–10 kDa range inhibits the formation of aristolactam‐DNA by 500‐fold, which could not be prevented by the addition of cofactors for SULTs and NATs. In conclusion, our results demonstrate that the genotoxicities of N‐hydroxyaristolactams depend on the cell type and involve not only sulfonation but also N,O‐acetyltransfer and an additional yet unknown mechanism(s). Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Triclosan at environmentally relevant concentrations promotes horizontal transfer of multidrug resistance genes within and across bacterial genera

BackgroundAntibiotic resistance poses an increasing threat to public health. Horizontal gene transfer (HGT) promoted by antibiotics is recognized as a significant pathway to disseminate antibiotic resistance genes (ARGs). However, it is unclear whether non-antibiotic, anti-microbial (NAAM) chemicals can directly promote HGT of ARGs in the environment. ObjectivesWe aimed to investigate whether triclosan (TCS), a widely-used NAAM chemical in personal care products, is able to stimulate the conjugative transfer of antibiotic multi-resistance genes carried by plasmid within and across bacterial genera. MethodsWe established two model mating systems, to investigate intra-genera transfer and inter-genera transfer. Escherichia coli K-12 LE392 carrying IncP-α plasmid RP4 was used as the donor, and E. coli K-12 MG1655 or Pseudomonas putida KT2440 were the intra- and inter-genera recipients, respectively. The mechanisms of the HGT promoted by TCS were unveiled by detecting oxidative stress and cell membrane permeability, in combination with Nanopore sequencing, genome-wide RNA sequencing and proteomic analyses. ResultsExposure of the bacteria to environmentally relevant concentrations of TCS (from 0.02 μg/L to 20 μg/L) significantly stimulated the conjugative transfer of plasmid-encoded multi-resistance genes within and across genera. The TCS exposure promoted ROS generation and damaged bacterial membrane, and caused increased expression of the SOS response regulatory genes umuC, dinB and dinD in the donor. In addition, higher expression levels of ATP synthesis encoding genes in E. coli and P. putida were found with increased TCS dosage. ConclusionsTCS could enhance the conjugative ARGs transfer between bacteria by triggering ROS overproduction at environmentally relevant concentrations. These findings improve our awareness of the hidden risks of NAAM chemicals on the spread of antibiotic resistance.

Development of a high‐throughput genotoxicity assay using Umu test strains expressing human cytochrome P450s and NADPH‐P450 reductase and bacterial O‐acetyltransferase

Umu test is one of the in vitro genotoxicity test that has been used widely. It was developed as a high-throughput test system using the 96-well microplate. We have previously constructed new umu test strains for the evaluation of genotoxicity of procarcinogenic metabolic products formed by cytochrome P450 (CYP) enzymes. In this study, a highly sensitive high-throughput genotoxicity test was developed using four umu test strains (OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4) that express human CYPs and NADPH-P450 reductase. We found that the modified umu-microplate method was more sensitive than the conventional microplate method using strain OY1002/1A2. In addition, the new microplate method was better able to detect genotoxicity than the test tube method when the strain OY1002/1A2 was used and had similar sensitivity for the remaining three strains. When the microplate method was used, OY1002/1A2 showed stronger umuC gene expression in the presence of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminofluorene, and 2-aminoanthracene compared to other strains. We also confirmed CYP1A2 expression in OY1002/1A2 in this condition. These results indicate that the microplate version of this test system can detect the genotoxicity of heterocyclic and aromatic amines with high sensitivity and can be used for high-throughput screening of potentially genotoxic compounds. Environ. Mol. Mutagen. 58:209-216, 2017. © 2017 Wiley Periodicals, Inc.

Evaluation of genotoxic effects of surface waters using a battery of bioassays indicating different mode of action

With the burgeoning contamination of surface waters threatening human health, the genotoxic effects of surface waters have received much attention. Because mutagenic and carcinogenic compounds in water cause tumors by different mechanisms, a battery of bioassays that each indicate a different mode of action (MOA) is required to evaluate the genotoxic effects of contaminants in water samples. In this study, 15 water samples from two source water reservoirs and surrounding rivers in Shijiazhuang city of China were evaluated for genotoxic effects. Target chemical analyses of 14 genotoxic pollutants were performed according to the Environmental quality standards for surface water of China. Then, the in vitro cytokinesis-block micronucleus (CBMN) assay, based on a high-content screening technique, was used to detect the effect of chromosome damage. The SOS/umu test using strain TA1535/pSK1002 was used to detect effects on SOS repair of gene expression. Additionally, two other strains, NM2009 and NM3009, which are highly sensitive to aromatic amines and nitroarenes, respectively, were used in the SOS/umu test to avoid false negative results. In the water samples, only two of the genotoxic chemicals listed in the water standards were detected in a few samples, with concentrations that were below water quality standards. However, positive results for the CBMN assay were observed in two river samples, and positive results for the induction of umuC gene expression in TA1535/pSK1002 were observed in seven river samples. Moreover, positive results were observed for NM2009 with S9 and NM3009 without S9 in some samples that had negative results using the strain TA1535/pSK1002. Based on the results with NM2009 and NM3009, some unknown or undetected aromatic amines and nitroarenes were likely in the source water reservoirs and the surrounding rivers. Furthermore, these compounds were most likely the causative pollutants for the genotoxic effect of these water samples. Therefore, to identify causative pollutants with harmful biological effects, chemical analyses for the pollutants listed in water quality standards is not sufficient, and single-endpoint bioassays may underestimate adverse effects. Thus, a battery of bioassays based on different MOAs is required for the comprehensive detection of harmful biological effects. In conclusion, for genotoxicity screening of surface waters, the SOS/umu test system by using different strains combined with the CBMN assay was a useful approach.

Identification of phototransformation products of the antiepileptic drug gabapentin: Biodegradability and initial assessment of toxicity

The anticonvulsant drug Gabapentin (GAB) is used for the treatment of various diseases (e.g. epilepsy, bipolar disorder, neuropathic pain) and is being consumed in high amounts. As GAB is not metabolized and shows a weak elimination in sewage treatment plants (STPs), it has been detected in surface water and even in raw potable water. Moreover, the confirmed teratogenic effects of GAB indicate the need for further investigations regarding options for the elimination of GAB in the water cycle. Little is known about the behavior of GAB during treatment with UV light, which is normally used for the disinfection of potable water and discussed for advanced wastewater treatment. In this study, GAB was exposed to polychromatic UV irradiation at different initial concentrations in aqueous solution. Afterwards the structures of the resulting phototransformation products (PTPs) were identified and elucidated by means of high-resolution mass spectrometry. GAB and photolytic mixtures were submitted to the Closed Bottle Test (CBT; OECD 301 D) to assess biodegradability. Furthermore, the toxicity of GAB and its photolytic mixtures was initially addressed on screening level using a modified luminescent bacteria test (LBT) and the umu-test (ISO/FDIS 13829). Environmentally realistic concentrations of GAB were disclosed by predicting STP influent concentrations (24.3 and 23.2 μg L−1). GAB with initial concentration of 100 mg L−1 was eliminated by 80% after 128 min of direct UV irradiation, but just 9% of non-purgeable organic carbon (NPOC) was removed indicating the formation of dead-end transformation products (TPs). Structures of different PTPs were elucidated and several identical PTPs could also be identified at lower initial treatment concentrations (20 mg L−1, 5 mg L−1, 1 mg L−1 and 0.1 mg L−1). GAB was classified as not readily biodegradable. Moreover, photo treatment did not result in better biodegradable PTPs. With increasing UV treatment duration, photolytic mixtures of GAB showed an increased inhibition of both, the bacterial luminescence emission as well as the growth in the modified LBT. In the umu-test no significant induction of the umuC gene as an indicator of genotoxicity was observed. Our results show that UV irradiation of GAB containing water would lead to the formation of recalcitrant PTPs. Considering that GAB was found in raw drinking water, the formation of toxic PTPs during drinking water treatment with UV light might be possible. Therefore, further studies should be conducted regarding the fate and effects on human health and the environment of GAB and the PTPs identified within this study.

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA- mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA- mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA- mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

Antibiotic Resistance Acquired through a DNA Damage-Inducible Response in Acinetobacter baumannii

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen that survives desiccation and quickly acquires resistance to multiple antibiotics. Escherichia coli gains antibiotic resistances by expressing genes involved in a global response to DNA damage. Therefore, we asked whether A. baumannii does the same through a yet undetermined DNA damage response akin to the E. coli paradigm. We found that recA and all of the multiple error-prone DNA polymerase V (Pol V) genes, those organized as umuDC operons and unlinked, are induced upon DNA damage in a RecA-mediated fashion. Consequently, we found that the frequency of rifampin-resistant (Rif(r)) mutants is dramatically increased upon UV treatment, alkylation damage, and desiccation, also in a RecA-mediated manner. However, in the recA insertion knockout strain, in which we could measure the recA transcript, we found that recA was induced by DNA damage, while uvrA and one of the unlinked umuC genes were somewhat derepressed in the absence of DNA damage. Thus, the mechanism regulating the A. baumannii DNA damage response is likely different from that in E. coli. Notably, it appears that the number of DNA Pol V genes may directly contribute to desiccation-induced mutagenesis. Sequences of the rpoB gene from desiccation-induced Rif(r) mutants showed a signature that was consistent with E. coli DNA polymerase V-generated base-pair substitutions and that matched that of sequenced A. baumannii clinical Rif(r) isolates. These data strongly support an A. baumannii DNA damage-inducible response that directly contributes to antibiotic resistance acquisition, particularly in hospitals where A. baumannii desiccates and tenaciously survives on equipment and surfaces.

Opposing effects of aminocoumarins and fluoroquinolones on the SOS response and adaptability in Staphylococcus aureus

RecA is the key enzyme involved in DNA repair, recombination and induction of the SOS response and is central to the development of antibiotic resistance. Here we assessed the interaction of two different gyrase inhibitors, ciprofloxacin (a fluoroquinolone) and novobiocin (an aminocoumarin), on RecA activity and the SOS response in Staphylococcus aureus. The influence of different gyrase inhibitors on the SOS response of S. aureus (including recA and lexA mutants) was analysed by northern blot analysis, real-time RT-PCR, western blot analysis and promoter activity assays. Recombination as well as mutation frequencies were determined for the different antibiotic combinations. We verified that ciprofloxacin leads to RecA activation and therefore induction of the SOS response. In contrast, novobiocin treatment resulted in an inhibition of recA transcription independent of LexA. When novobiocin and ciprofloxacin were added simultaneously, recA was reduced to the same level as with novobiocin alone. In combination, novobiocin also partially reduces the ciprofloxacin-mediated induction of the LexA target gene umuC (error-prone polymerase). Apart from reducing recA and umuC expression, novobiocin also inhibited the frequency of recombination, mutation and the formation of non-haemolytic variants. In summary, aminocoumarins inhibit recA expression in S. aureus and probably delay the process of developing antibiotic resistance and gene transfer. A clinical re-evaluation of these compounds as well as designing more applicable derivatives should be considered.

Production of a new terpenoid from biotransformation of (-)-isolongifolanol by Aspergillus niger and suppression of SOS-inducing activity

The stereoselective oxidation of (—)-isolongifolanol (1) with a longifolene skeleton by Aspergillus niger (NBRC 4414) as a biocatalyst and suppressive effect on umuC gene expression by chemical mutagens furylfuramid and AFB1 of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Compound 1 was converted to a new terpenoid, (-)-(2S,8R)-8,12-dihydroxy-isolongifolanol (2). Its structure was determined by NMR, IR, specific rotation and mass spectrometry. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB1 in the umu test. Compound 1 suppressed 51% of the SOS-inducing activity against furylfuramid at < 1.0 mM. Compound 2 suppressed 15% and 24% of the SOS-inducing activity against furylfuramid and AFB1 at < 1.0 mM respectively.

Suppression of SOS-Inducing Activity of Chemical Mutagens by Metabolites from Microbial Transformation of (+)-Longicyclene

In this study, biotransformation of (+)-longicyclene (1) by Aspergillus niger (NBRC 4414) and the suppressive effect on umuC gene expression by chemical mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and aflatoxin B1 (AFB1) of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Initially, compound 1 was converted to three new terpenoids, (-)-(10R)-10-hydroxy-longicyclic acid (2), (+)-(10S)-10-hydroxy-longicyclic acid (3), and (+)-10-oxo-longicyclic acid (4) by A. niger , and their conversion rates were 27, 23, and 30%, respectively. The metabolites suppressed the SOS-inducing activity of furylfuramide and AFB1 in the umu test. Compounds 1-4 were hardly showing a suppressive effect on umu gene expression of the SOS responses in S. typhimurium TA1535/pSK1002 against furylfuramid. However, metabolites showed a suppressive effect against AFB1. Compound 4 had gene expression by chemical mutagen AFB1, was suppressed 53% at <1.0 mM, and was the most effective compound in this experiment.

Suppression of SOS-Inducing Activity of Chemical Mutagens by Metabolites from Microbial Transformation of (−)-Isolongifolene

In this study, biotransformation of (-)-isolongifolene (1) by Glomerella cingulata and suppressive effect on umuC gene expression by chemical mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and aflatoxin B(1) (AFB(1)) of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Initially, 1 was carried out the microbial transformation by G. cingulata. The result found that 1 was converted into (-)-isolongifolen-9-one (2), (-)-(2S)-13-hydroxy-isolongifolen-9-one (3), and (-)-(4R)-4-hydroxy-isolongifolen-9-one (4) by G. cingulata, and their conversion rates were 60, 25, and 15%, respectively. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB(1) in the umu test. Comound 2 showed gene expression by chemical mutagens furylfuramide and AFB(1) was suppressed 54 and 50% at <0.5 mM, respectively. Compound 2 is the most effective compound in this experiment.

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimurium umu strains expressing human cytochrome P450 and N-acetyltransferase

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant ( Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 ( O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.

Genotoxic Activation of the Environmental Pollutant 3-Nitrobenzanthrone by Human Cytochrome P450 Enzymes Expressed in Salmonella typhimurium umu Tester Strains

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic compound identified in diesel exhaust, airborne particulate matter, soil, and water. To address whether 3-NBA shows genotoxic effects through any metabolic activation pathway, we determined the genotoxicity of 3-NBA using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. In order to further clarify the role of human cytochrome P450 (P450 or CYP) enzyme in the bioactivation of 3-NBA to genotoxic metabolites, umu tester strains S. typhimurium OY1022/1A1, OY1022/1A2, OY1022/1B1, and OY1022/3A4, which express four respective human cytochrome P450 enzymes, NADPH-cytochrome P450 reductases (NPR) and O-AT were established by introducing two plasmids into S. typhimurium TA1535NR/1,8-DNP (nitroreductase-deficient and resistant to 1,8-dinitropyrene, probably due to functional loss of O-AT), one carrying both P450 and reductase cDNA in a biocistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC'-'lacZ fusion genes. Induction of umuC gene expression could be monitored by measuring the cellular β-galactosidase activity produced by the umuC'-'lacZ fusion gene. Although the induction of umuC by 3-NBA was weak in TA1535/pSK1002, NM1000, and NM2000 strains, the induction was considerably potent in NM2009 and NM3009 strains. 3-NBA was also found to induce umuC gene expression in OY1022/1A1 and OY1022/3A4 strains and, to a lesser extent, OY1022/1A2 and OY1022/1B1 strains, at a much higher rate than the parental OY1022/pCW strain. Collectively, these results suggest nitroreduction and O-acetylation by bacterial NR and O-AT, respectively, and nitroreduction by human CYP1A1, 3A4, 1A2, and 1B1 and NPR could contribute to the genotoxic activation of 3-NBA to produce reactive metabolites.

Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.