Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA.

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

Comparative Genomic Analysis of Cold-Water Coral-Derived Sulfitobacter faviae: Insights into Their Habitat Adaptation and Metabolism.

Sulfitobacter is one of the major sulfite-oxidizing alphaproteobacterial groups and is often associated with marine algae and corals. Their association with the eukaryotic host cell may have important ecological contexts due to their complex lifestyle and metabolism. However, the role of Sulfitobacter in cold-water corals remains largely unexplored. In this study, we explored the metabolism and mobile genetic elements (MGEs) in two closely related Sulfitobacter faviae strains isolated from cold-water black corals at a depth of ~1000 m by comparative genomic analysis. The two strains shared high sequence similarity in chromosomes, including two megaplasmids and two prophages, while both contained several distinct MGEs, including prophages and megaplasmids. Additionally, several toxin-antitoxin systems and other types of antiphage elements were also identified in both strains, potentially helping Sulfitobacter faviae overcome the threat of diverse lytic phages. Furthermore, the two strains shared similar secondary metabolite biosynthetic gene clusters and genes involved in dimethylsulfoniopropionate (DMSP) degradation pathways. Our results provide insight into the adaptive strategy of Sulfitobacter strains to thrive in ecological niches such as cold-water corals at the genomic level.

Comparative genomic insights into habitat adaptation of coral-associated Prosthecochloris.

Green sulfur bacteria (GSB) are a distinct group of anoxygenic phototrophic bacteria that are found in many ecological niches. Prosthecochloris, a marine representative genus of GSB, was found to be dominant in some coral skeletons. However, how coral-associated Prosthecochloris (CAP) adapts to diurnal changing microenvironments in coral skeletons is still poorly understood. In this study, three Prosthecochloris genomes were obtained through enrichment culture from the skeleton of the stony coral Galaxea fascicularis. These divergent three genomes belonged to Prosthecochloris marina and two genomes were circular. Comparative genomic analysis showed that between the CAP and non-CAP clades, CAP genomes possess specialized metabolic capacities (CO oxidation, CO2 hydration and sulfur oxidation), gas vesicles (vertical migration in coral skeletons), and cbb 3-type cytochrome c oxidases (oxygen tolerance and gene regulation) to adapt to the microenvironments of coral skeletons. Within the CAP clade, variable polysaccharide synthesis gene clusters and phage defense systems may endow bacteria with differential cell surface structures and phage susceptibility, driving strain-level evolution. Furthermore, mobile genetic elements (MGEs) or evidence of horizontal gene transfer (HGT) were found in most of the genomic loci containing the above genes, suggesting that MGEs play an important role in the evolutionary diversification between CAP and non-CAP strains and within CAP clade strains. Our results provide insight into the adaptive strategy and population evolution of endolithic Prosthecochloris strains in coral skeletons.

Antitoxin CrlA of CrlTA Toxin-Antitoxin System in a Clinical Isolate Pseudomonas aeruginosa Inhibits Lytic Phage Infection.

Pseudomonas aeruginosa is an important opportunistic pathogen in cystic fibrosis patients and immunocompromised individuals, and the toxin-antitoxin (TA) system is involved in bacterial virulence and phage resistance. However, the roles of TA systems in P. aeruginosa are relatively less studied and no phage Cro-like regulators were identified as TA components. Here, we identified and characterized a chromosome-encoded prophage Cro-like antitoxin (CrlA) in the clinical isolate P. aeruginosa WK172. CrlA neutralized the toxicity of the toxin CrlA (CrlT) which cleaves mRNA, and they formed a type II TA system. Specifically, crlA and crlT are co-transcribed and their protein products interact with each other directly. The autorepression of CrlA is abolished by CrlT through the formation of the CrlTA complex. Furthermore, crlTA is induced in the stationary phase, and crlA is expressed at higher levels than crlT. The excess CrlA inhibits the infection of lytic Pseudomonas phages. CrlA is widely distributed among Pseudomonas and in other bacterial strains and may provide antiphage activities.

Filamentous prophage capsid proteins contribute to superinfection exclusion and phage defence in Pseudomonas aeruginosa.

Filamentous prophages in Pseudomonas aeruginosa PAO1 are converted to superinfective phage virions during biofilm development. Superinfection exclusion is necessary for the development of resistance against superinfective phage virions in host cells. However, the molecular mechanisms underlying the exclusion of superinfective Pf phages are unknown. In this study, we found that filamentous prophage-encoded structural proteins allow exclusion of superinfective Pf phages by interfering with type IV pilus (T4P) function. Specifically, the phage minor capsid protein pVII inhibits Pf phage adsorption by interacting with PilC and PilJ of T4P, and overproduction of pVII completely abrogates twitching motility. The minor capsid protein pIII provides partial superinfection exclusion and interacts with the PilJ and TolR/TolA proteins. Furthermore, pVII provides full host protection against infection by pilus-dependent lytic phages, and pIII provides partial protection against infection by pilus-independent lytic phages. Considering that filamentous prophages are common in clinical Pseudomonas isolates and their induction is often activated during biofilm formation, this study suggests the need to rethink the strategy of using lytic phages to treat P. aeruginosa biofilm-related infections.

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment.

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.

Rapid detection of temperate bacteriophage using a simple motility assay.

Phage contamination is a common complication for the fermentation and pharmaceutical industries. The risk of bacteriophage contamination in laboratory processes increases with multiple rounds of genetic manipulation such as deletion and complementation. The contamination of temperate phages does not lead to immediate host cell lysis but could become a serious issue when the lytic cycle is activated under specific conditions. Our objective was to develop a quick and reliable detection method for checking possible temperate phage contamination. Here, using motility plates, we found that when the strain carries a newly acquired temperate phage, its presence can be easily detected by the formation of a clear 'lysis zone' when swimming against the original strain on the same swimming plates. Compared to the traditional double agar layer method and genomic sequencing-based methods, the duration of the motility-based assay is shorter and the procedure is simplified. More importantly, for the bacterial strains that already contain active prophages, this method can still easily detect the newly acquired phages without tedious phage identification procedure. These features make this method highly applicable to laboratory and industrial processes.

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella.

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.

Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system.

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa.

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5'-AATTCN5 GTTAA-3') overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.

OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Characterization of Two Toxin-Antitoxin Systems in Deep-Sea Streptomyces sp. SCSIO 02999.

Toxin-antitoxin (TA) systems are ubiquitous and abundant genetic elements in bacteria and archaea. Most previous TA studies have focused on commensal and pathogenic bacteria, but have rarely focused on marine bacteria, especially those isolated from the deep sea. Here, we identified and characterized three putative TA pairs in the deep-sea-derived Streptomyces sp. strain SCSIO 02999. Our results showed that Orf5461/Orf5462 and Orf2769/Orf2770 are bona fide TA pairs. We provide several lines of evidence to demonstrate that Orf5461 and Orf5462 constitute a type-II TA pair that are homologous to the YoeB/YefM TA pair from Escherichia coli. Although YoeB from SCSIO 02999 was toxic to an E. coli host, the homologous YefM antitoxin from SCSIO 02999 did not neutralize the toxic effect of YoeB from E. coli. For the Orf2769/Orf2770 TA pair, Orf2769 overexpression caused significant cell elongation and could lead to cell death in E. coli, and the neighboring Orf2770 could neutralize the toxic effect of Orf2769. However, no homologous toxin or antitoxin was found for this pair, and no direct interaction was found between Orf2769 and Orf2770. These results suggest that Orf2769 and Orf2770 may constitute a novel TA pair. Thus, deep-sea bacteria harbor typical and novel TA pairs. The biochemical and physiological functions of different TAs in deep-sea bacteria warrant further investigation.

Phages Mediate Bacterial Self-Recognition.

Cells are social, and self-recognition is a conserved aspect of group behavior where cells assist kin and antagonize non-kin. However, the role of phage in self-recognition is unexplored. Here we find that a demarcation line is formed between different swimming Escherichia coli strains but not between identical clones; hence, motile cells discriminate between self and non-self. The basis for this self-recognition is a 49 kb, T1-type, lytic phage of the family Siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the host's cryptic prophage proteins, YfdM of CPS-53, to propagate. Critically, SW1 provides a conditional benefit to E. coli K-12 compared with the identical strain that lacks the phage. A demarcation line is also formed when strains harbor either the lysogenic phage ϕ80 or lambda and encounter siblings that lack the lysogen. In summary, bacteria can use phage to distinguish siblings that lack phage.

Antitoxin HigA inhibits virulence gene mvfR expression in Pseudomonas aeruginosa.

Toxin/antitoxin (TA) systems are ubiquitous in bacteria and archaea and participate in biofilm formation and stress responses. The higBA locus of the opportunistic pathogen Pseudomonas aeruginosa encodes a type II TA system. Previous work found that the higBA operon is cotranscribed and that HigB toxin regulates biofilm formation and virulence expression. In this study, we demonstrate that HigA antitoxin is produced at a higher level than HigB and that higA mRNA is expressed separately from a promoter inside higB during the late stationary phase. Critically, HigA represses the expression of mvfR, which is an important virulence-related regulator, by binding to a conserved HigA palindrome (5'-TTAAC GTTAA-3') in the mvfR promoter, and the binding of HigB to HigA derepresses this process. During the late stationary phase, excess HigA represses the expression of mvfR and higBA. However, in the presence of aminoglycoside antibiotics where Lon protease is activated, the degradation of HigA by Lon increases P. aeruginosa virulence by simultaneously derepressing mvfR and higB transcription. Therefore, this study reveals that the antitoxin of the P. aeruginosa TA system is integrated into the key virulence regulatory network of the host and functions as a transcriptional repressor to control the production of virulence factors.

Resistance to oxidative stress by inner membrane protein ElaB is regulated by OxyR and RpoS.

C-tail anchored inner membrane proteins are a family of proteins that contain a C-terminal transmembrane domain but lack an N-terminal signal sequence for membrane targeting. They are widespread in eukaryotes and prokaryotes and play critical roles in membrane traffic, apoptosis and protein translocation in eukaryotes. Recently, we identified and characterized in Escherichia coli a new C-tail anchored inner membrane, ElaB, which is regulated by the stationary phase sigma factor RpoS. ElaB is important for resistance to oxidative stress but the exact mechanism is unclear. Here, we show that ElaB functions as part of the adaptive oxidative stress response by maintaining membrane integrity. Production of ElaB is induced by oxidative stress at the transcriptional level. Moreover, elaB expression is also regulated by the key regulator OxyR via an OxyR binding site in the promoter of elaB. OxyR induces the expression of elaB in the exponential growth phase, while excess OxyR reduces elaB expression in an RpoS-dependent way in the stationary phase. In addition, deletion of elaB reduced fitness compared to wild-type cells after prolonged incubation. Therefore, we determined how ElaB is regulated under oxidative stress: RpoS and OxyR coordinately control the expression of inner membrane protein ElaB.

Colistin Resistance Gene mcr-1 Mediates Cell Permeability and Resistance to Hydrophobic Antibiotics.

Colistin is considered the last-resort antibiotic used to treat multidrug resistant bacteria-related infections. However, the discovery of the plasmid-mediated colistin resistance gene, mcr-1, threatens the clinical utility of colistin antibiotics. In this study, the physiological function of MCR-1, which encodes an LPS-modifying enzyme, was investigated in E. coli K-12. Specifically, the impact of mcr-1 on membrane permeability and antibiotic resistance of E. coli was assessed by constructing an mcr-1 deletion mutant and by a complementation study. The removal of the mcr-1 gene from plasmid pHNSHP45 not only led to reduced resistance to colistin but also resulted in a significant change in the membrane permeability of E. coli. Unexpectedly, the removal of the mcr-1 gene increased cell viability under high osmotic stress conditions (e.g., 7.0% NaCl) and led to increased resistance to hydrophobic antibiotics. Increased expression of mcr-1 also resulted in decreased growth rate and changed the cellular morphology of E. coli. Collectively, our results revealed that the spread of mcr-1-carrying plasmids alters other physiological functions in addition to conferring colistin resistance.

Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa.

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis-lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5'-untranslated regions overlap. XisF4 and Pf4r not only auto-activate their own expression but also repress each other. Furthermore, two H-NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.

Antimicrobial Resistance Profile of mcr-1 Positive Clinical Isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

Type II toxin/antitoxin system ParESO /CopASO stabilizes prophage CP4So in Shewanella oneidensis.

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.