SOS Response Research Articles

Electrochemical disinfection may increase the spread of antibiotic resistance genes by promoting conjugal plasmid transfer

Current in the milliampere range can be used for electrochemical inactivation of bacteria. Yet, bacteria-including antibiotic resistant bacteria (ARB) may be subjected to sublethal conditions due to imperfect mixing or energy savings measures during electrochemical disinfection. It is not known whether such sublethal current intensities have the potential to stimulate plasmid transfer from ARB. In this study, conjugal transfer of plasmid pKJK5 was investigated between Pseudomonas putida strains under conditions reflecting electrochemical disinfection. Although the abundance of culturable and membrane-intact donor and recipient cells decreased with applied current (0–60 mA), both transconjugant density and transconjugant frequency increased. Both active chlorine and superoxide radicals were generated electrolytically, and ROS generation was induced. In addition, we detected significant over expression of a core oxidative stress defense gene (ahpCF) with current. Expression of selected conjugation related genes (traE, traI, trbJ, and trbL) also significantly correlated with current intensity. ROS accumulation, SOS response and subsequent derepression of conjugation are therefore the plausible consequence of sublethal current exposure. These findings suggest that sublethal intensities of current can enhance conjugal plasmid transfer, and that it is essential that conditions of electrochemical disinfection (applied voltage, current density, time and mixing) are carefully controlled to avoid conjugal ARG transmission.

Horizontal transfer of intracellular and extracellular ARGs in sludge compost under sulfamethoxazole stress

Aerobic composting has been considered a safe way to recycle sludge, providing a green recycling system that conserves resources. Sludge from sewage treatment plants has been identified as a significant reservoir for ARGs. However, it remains unclear whether the residual antibiotics in sludge affect the horizontal transfer mechanism of intracellular and extracellular antibiotic resistance genes (iARGs, eARGs) in compost. Here, intracellular, extracellular and total genomic DNA was extracted during sulfamethoxazole (SMX) selective pressure sludge composting. After composting, most of the sul in the sludge was removed, the removal rate of iARGs and eARGs was around 80 % and 90 %. The presence of antibiotic stress in sludge would promote the horizontal transfer of ARGs. Metagenomic sequencing confirmed that the sul gene cluster was carried by multiple mobile genetic elements, such as the Tn3 transposon and intl1, which promoted the horizontal transfer of iARGs. SMX selective pressure increased the expression of genes responsible for SOS response (29.52 %), type IV secretion systems (28.09 %) and type IV pili (40.45 %). Combined with the KEGG annotation analysis, SMX facilitated intake to eARGs by hosts, such as Pseudomonas, via type IV pili. Overall, this is the first study to verify that the horizontal transfer of iARGs and eARGs is promoted by the presence of antibiotic residues in sludge during composting.

Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs).

Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.

Triclosan Promotes Conjugative Transfer of Antibiotic Resistance Genes to Opportunistic Pathogens in Environmental Microbiome.

Although triclosan, as a widely used antiseptic chemical, is known to promote the transmission of antibiotic resistance to diverse hosts in pure culture, it is still unclear whether and how triclosan could affect the transmission of broad-host-range plasmids among complex microbial communities. Here, bacterial culturing, fluorescence-based cell sorting, and high-throughput 16S rRNA gene amplicon sequencing were combined to investigate contributions of triclosan on the transfer rate and range of an IncP-type plasmid from a proteobacterial donor to an activated sludge microbiome. Our results demonstrate that triclosan significantly enhances the conjugative transfer of the RP4 plasmid among activated sludge communities at environmentally relevant concentrations. High-throughput 16S rRNA gene sequencing on sorted transconjugants demonstrates that triclosan not only promoted the intergenera transfer but also the intragenera transfer of the RP4 plasmid among activated sludge communities. Moreover, triclosan mediated the transfer of the RP4 plasmid to opportunistic human pathogens, for example, Legionella spp. The mechanism of triclosan-mediated conjugative transfer is primarily associated with excessive oxidative stress, followed by increased membrane permeability and provoked SOS response. Our findings offer insights into the impacts of triclosan on the dissemination of antibiotic resistance in the aquatic environmental microbiome.

Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.

Antibacterial T6SS effectors with a VRR-Nuc domain are structure-specific nucleases.

The type VI secretion system (T6SS) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here, we characterize the function of the SPI-22 T6SS of Salmonella bongori showing that it has antibacterial activity and identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins with a DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV3 recognizes specific DNA structures and preferentially cleave splayed arms, generating DNA double-strand breaks and inducing the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanisms regulating the activity of these toxins.

Locking down SOS Mutagenesis Repression in a Dynamic Pathogen.

The article "The DdrR coregulator of the Acinetobacter baumannii mutagenic DNA damage response potentiates UmuDAb repression of error-prone polymerases" in this issue of the J Bacteriol, (D. Cook, M. D. Flannigan, B. V. Candra, K. D. Compton, and J. M. Hare., J Bacteriol 204:e00165-22, 2022, https://doi.org/10.1128/jb.00165-22) reveals a more detailed understanding of the regulatory mechanism of the SOS response in Acinetobacter baumannii. This information provides novel targets for development of antimicrobial therapies against this ESKAPE pathogen and new insight into the complex regulation of the SOS stress-response.

Resistance of Bacteria toward 475 nm Blue Light Exposure and the Possible Role of the SOS Response.

The increase in antibiotic resistance represents a major global challenge for our health systems and calls for alternative treatment options, such as antimicrobial light-based therapies. Blue light has shown promising results regarding the inactivation of a variety of microorganisms; however, most often, antimicrobial blue light (aBL) therapy is performed using wavelengths close to the UV range. Here we investigated whether inactivation was possible using blue light with a wavelength of 475 nm. Both Gram-positive and -negative bacterial strains were treated with blue light with fluences of 7.5–45 J/cm2. Interestingly, only some bacterial strains were susceptible to 475 nm blue light, which was associated with the lack of RecA, i.e., a fully functional DNA repair mechanism. We demonstrated that the insertion of the gene recA reduced the susceptibility of otherwise responsive bacterial strains, indicating a protective mechanism conveyed by the bacterial SOS response. However, mitigating this pathway via three known RecA inhibiting molecules (ZnAc, curcumin, and Fe(III)-PcTs) did not result in an increase in bactericidal action. Nonetheless, creating synergistic effects by combining a multitarget therapy, such as aBL, with an RecA targeting treatment could be a promising strategy to overcome the dilemma of antibiotic resistance in the future.

Defects in DNA double-strand break repair resensitize antibiotic-resistant Escherichia colito multiple bactericidal antibiotics.

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance‐breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double‐strand break repair as a promising target for the development of resistance‐breaking co‐therapies. We examined genetic variants of Escherichia coli that combined antibiotic‐resistance determinants with DNA repair defects. We observed that defects in the double‐strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double‐strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break‐repair inhibitors can be developed they could resensitize antibiotic‐resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic‐resistance mutations.

Fe2+ protects postharvest pitaya (Hylocereus undulatus britt) from Aspergillus. flavus infection by directly binding its genomic DNA

Aspergillus flavus (A. flavus) is a postharvest fungus, causing pitaya fruit decay and limiting pitaya value and shelf life. However, safer and more efficient methods for preventing A. flavus contamination for pitaya fruit remain to be investigated. In this study, we successfully proved exogenous Fe2+ could inhibit A. flavus colonization in pitaya fruit and extend pitaya’s shelf life after harvest. Moreover, gel electrophoresis, CD analysis and Raman spectrum tests revealed Fe2+ could more effectively and thoroughly promote conidial death by directly binding to A. flavus DNA. Increased expression of DNA damage repair-related genes after Fe2+ treatment was observed by transcription analysis, which might eventually lead to SOS response in A. flavus. These results indicated Fe2+ could prevent A. flavus infestation on pitaya in a novel, quickly responsive mechanism. Our results shed light on the potential application of Fe2+ in the food industry and provided a more universal antifungal agent against food pathogens.

Direct visualization of translesion DNA synthesis polymerase IV at the replisome

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses, we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the β clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the β clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS− bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA.

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ’s attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

Unraveling the impact and mechanism of antipyretic paracetamol on intergenera conjugative plasmid transfer

Antimicrobial resistance has been considered as a great threat to biosecurity and human health. And the transmission of antibiotic resistance genes (ARGs) by conjugated plasmid is a key factor in the prevalence of antimicrobial resistance. Paracetamol (PRC), one of nonopioid analgesics, is an extensively used antipyretic and mild analgesic worldwide available for numerous prescriptions. It was unclear whether PRC could promote the spread of ARGs. Here, it was demonstrated that PRC promoted intergenera conjugative plasmid transfer in an established conjugation model. Both donor and recipient strains treated by PRC emerged the variations of reactive oxygen species (ROS), SOS response and cell membrane permeability. Correspondingly, transcriptome analysis revealed that the gene expression involved in cell membrane permeability and SOS response was up-regulated significantly after PRC exposure. More directly, PRC also increased the expressions of conjugation related genes of trbG and trbP in donor. This study proved for the first time that PRC could enhance the intergenera conjugative plasmid transfer. Collectively, these findings manifested the potential threat associated with the existence of non-antibiotic substance PRC, which could provide an important insight into antimicrobial resistance spread.

Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer.

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. Bacteria deploy SIM when faced with DNA double-strand breaks in the presence of conditions that elicit an SOS response. SIM employs DinB, the evolutionary precursor to human trans-lesion synthesis (TLS) error-prone polymerases, and results in mutations concentrated around DNA double-strand breaks with an abundance that decays with distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors, non-inherited mutations from 129 normal samples, and acquired mutations in 3 cell line models of stress-induced adaptive mutation. We introduce statistical methods to identify mutational clusters, quantify their shapes and tease out the potential mechanism that produced them. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. Clusters in normal samples occur more often in the same genomic location across samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis. Additionally, the signatures of TLS contribute the most to mutational cluster formation in both patient samples as well as experimental models of SIM. Furthermore, a measure of cluster shape heterogeneity was associated with cancer patient survival with a hazard ratio of 5.744 (Cox Proportional Hazard Regression, 95% CI: 1.824–18.09). Our results support the conclusion that the ancient and evolutionary-conserved adaptive mutation response found in bacteria is a source of genomic instability in cancer. Biological adaptation through SIM might explain the ability of tumors to evolve in the face of strong selective pressures such as treatment and suggests that the conventional ‘hit it hard’ approaches to therapy could prove themselves counterproductive.

Effects of functional modules and bacterial clusters response on transmission performance of antibiotic resistance genes under antibiotic stress during anaerobic digestion of livestock wastewater

The formation and transmission of antibiotic resistance genes (ARGs) have attracted increasing attention. It is unclear whether the internal mechanisms by which antibiotics affect horizontal gene transfer (HGT) of ARGs during anaerobic digestion (AD) were influenced by dose and type. We investigated the effects of two major antibiotics (oxytetracycline, OTC, and sulfamethoxazole, SMX) on ARGs during AD according to antibiotic concentration in livestock wastewater influent. The low-dose antibiotic (0.5 mg/L) increased ROS and SOS responses, promoting the formation of ARGs. Meanwhile, low-dose antibiotics could also promote the spread of ARGs by promoting pili, communication responses, and the type IV secretion system (T4SS). However, different types and doses of antibiotics would lead to changes in the above functional modules and then affect the enrichment of ARGs. With the increasing dose of SMX, the advantages of pili and communication responses would gradually change. In the OTC system, low-dose has the strongest promoting ability in both pili and communication responses. Similarly, an increase in the dose of SMX would change T4SS from facilitation to inhibition, while OTC completely inhibits T4SS. Microbial and network analysis also revealed that low-dose antibiotics were more favorable for the growth of host bacteria.

A neglected risk of nanoplastics as revealed by the promoted transformation of plasmid‐borne ampicillin resistance gene by Escherichia coli

Plastic pollution and antibiotic resistance are two emerging environmental and human health crises today. Although it was revealed that microplastics can serve as vectors for the dissemination of antibiotic resistance, it is still unclear how the nanoplastics influence the horizontal transfer of antibiotic resistance genes (ARGs). Herein, we firstly compared the effect of polystyrene (PS) micro/nanoplastics on the transformation of plasmid-borne ARG, using a transformation model consisting of plasmid pUC19 (ampR ) and Escherichia coli DH5α (recipient). Due to its size effect, PS nanoplastics (10-500 mg/L) significantly enhanced the transformation efficiency (2.8-5.4 folds) and frequency (3.2-8.4 folds) of exogenous ampR into E. coli, while PS microplastics exerted no influence. The detailed mechanisms were found that nanoplastics induced reactive oxygen species (ROS) overproduction, activated SOS response, increased cell membrane permeability and changed the secretion systems, thereby facilitating the uptake of exogenous DNA by bacteria. Moreover, the co-presences of nanoplastics with humic acid or Fe3+ relieved to some extent, but did not completely alleviate the promoting effect of nanoplastics on plasmid transformation. Our findings suggest that the risk of nanoplastics on promoting the dissemination of antibiotic resistance should not be neglected, and further studies are needed to investigate such risk in complex environments.

Antidepressants promote the spread of antibiotic resistance via horizontally conjugative gene transfer.

Antibiotic resistance is a global concern threatening public health. Horizontal gene transfer (HGT) between bacterial species contributes greatly to the dissemination of antibiotic resistance. Conjugation is one of the major HGT pathways responsible for the spread of antibiotic resistance genes (ARGs). Antidepressant drugs are commonly prescribed antipsychotics for major depressive disorders and are frequently detected in aquatic environments. However, little is known about how antidepressants stress bacteria and whether such effect can promote conjugation. Here, we report that commonly prescribed antidepressants, sertraline, duloxetine, fluoxetine, and bupropion, can promote the conjugative transfer of plasmid-borne multidrug resistance genes carried by environmentally and clinically relevant plasmids. Noteworthy, the transfer of plasmids across bacterial genera is significantly enhanced by antidepressants at clinically relevant concentrations. We also reveal the underlying mechanisms of enhanced conjugative transfer by employing flow cytometric analysis, genome-wide RNA sequencing and proteomic analysis. Antidepressants induce the production of reactive oxygen species and the SOS response, increase cell membrane permeability, and upregulate the expression of conjugation relevant genes. Given the contribution of HGT in the dissemination of ARGs, our findings highlight the importance of prudent prescription of antidepressants and to the potential connection between antidepressants and increasing antibiotic resistance.

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Insights into the microbial response mechanisms to ciprofloxacin during sulfur-mediated biological wastewater treatment using a metagenomics approach

The fate and removal of ciprofloxacin, a class of fluoroquinolone antibiotic, during sulfur-mediated biological wastewater treatment has been recently well documented. However, little is known regarding the genetic response of microorganisms to ciprofloxacin. Here, a lab-scale anaerobic sulfate-reducing bioreactor was continuously operated over a long term for ciprofloxacin-contaminated wastewater treatment to investigate the response of the microorganisms to ciprofloxacin by adopting a metagenomics approach. It was found that total organic carbon (TOC) removal and sulfate reduction were promoted by approximately 10% under ciprofloxacin stress, along with the enrichment of functional genera (e.g., Desulfobacter, Geobacter) involved in carbon and sulfur metabolism. The metagenomic analytical results demonstrated that ciprofloxacin triggered the microbial SOS response, as demonstrated by the up-regulation of the multidrug efflux pump genes (8–125-fold higher than that of the control) and ciprofloxacin-degrading genes (4–33-fold higher than that of the control). Moreover, the contents of ATP, NADH, and cytochrome C, as well as related functional genes (including genes involved in energy generation, electron transport, carbon metabolism, and sulfur metabolism) were markedly increased under ciprofloxacin stress. This demonstrated that the carbon and sulfur metabolisms were enhanced for energy (ATP) generation and electron transport in response to ciprofloxacin-induced stress. Interestingly, the microbes tended to cooperate while being subjected exposure to exogenous ciprofloxacin according to the reconstructed metabolic network using the NetSeed model. Particularly, the species with higher complementarity indices played more pivotal roles in strengthening microbial metabolism and the SOS response under long-term ciprofloxacin stress. This study characterized the response mechanisms of microorganisms to ciprofloxacin at the genetic level in sulfur-mediated biological wastewater treatment. These new understandings will contribute the scientific basis for improving and optimizing the sulfur-mediated bioprocess for antibiotics-laden wastewater treatment.