Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system.

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO-K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

The HipAB Toxin-Antitoxin System Stabilizes a Composite Genomic Island in Shewanella putrefaciens CN-32.

Composite genomic islands (GIs) are useful models for studying GI evolution if they can revert into the previous components. In this study, CGI48-a 48,135-bp native composite GI that carries GI21, whose homologies specifically integrated in the conserved yicC gene-were identified in Shewanella putrefaciens CN-32. CGI48 was integrated into the tRNATrp gene, which is a conserved gene locus for the integration of genomic islands in Shewanella. Upon expressing integrase and excisionase, CGI48 and GI21 are excised from chromosomes via site-specific recombination. The shorter attachment sites of GI21 facilitated the capture of GI21 into CGI48. Moreover, GI21 encodes a functional HipAB toxin-antitoxin system, thus contributing to the maintenance of CGI48 in the host bacteria. This study provides new insights into GI evolution by performing the excision process of the inserting GI and improves our understanding of the maintenance mechanisms of composite GI.

App based education programme to reduce salt intake (AppSalt) in schoolchildren and their families in China: parallel, cluster randomised controlled trial.

OBJECTIVE: To determine whether a smartphone application based education programme can lower salt intake in schoolchildren and their families. DESIGN: Parallel, cluster randomised controlled trial, with schools randomly assigned to either intervention or control group (1:1). SETTING: 54 primary schools from three provinces in northern, central, and southern China, from 15 September 2018 to 27 December 2019. PARTICIPANTS: 592 children (308 (52.0%) boys; mean age 8.58 (standard deviation 0.41) years) in grade 3 of primary school (about 11 children per school) and 1184 adult family members (551 (46.5%) men; mean age 45.80 (12.87) years). INTERVENTION: Children in the intervention group were taught, with support of the app, about salt reduction and assigned homework to encourage their families to participate in activities to reduce salt consumption. MAIN OUTCOME MEASURES: Primary outcome was the difference in salt intake change (measured by 24 hour urinary sodium excretion) at 12 month follow-up, between the intervention and control groups. RESULTS: After baseline assessment, 297 children and 594 adult family members (from 27 schools) were allocated to the intervention group, and 295 children and 590 adult family members (from 27 schools) were allocated to the control group. During the trial, 27 (4.6%) children and 112 (9.5%) adults were lost to follow-up, owing to children having moved to another school or adults unable to attend follow-up assessments. The remaining 287 children and 546 adults (from 27 schools) in the intervention group and 278 children and 526 adults (from 27 schools) in the control group completed the 12 month follow-up assessment. Mean salt intake at baseline was 5.5 g/day (standard deviation 1.9) in children and 10.0 g/day (3.5) in adults in the intervention group, and 5.6 g/day (2.1) in children and 10.0 g/day (3.6) in adults in the control group. During the study, salt intake of the children increased in both intervention and control groups but to a lesser extent in the intervention group (mean effect of intervention after adjusting for confounding factors -0.25 g/day, 95% confidence interval -0.61 to 0.12, P=0.18). In adults, salt intake decreased in both intervention and control groups but to a greater extent in the intervention group (mean effect -0.82 g/day, -1.24 to -0.40, P<0.001). The mean effect on systolic blood pressure was -0.76 mm Hg (-2.37 to 0.86, P=0.36) in children and -1.64 mm Hg (-3.01 to -0.27, P=0.02) in adults. CONCLUSIONS: The app based education programme delivered through primary school, using a child-to-parent approach, was effective in lowering salt intake and systolic blood pressure in adults, but the effects were not significant in children. Although this novel approach could potentially be scaled up to larger populations, the programme needs further strengthening to reduce salt intake across the whole population, including schoolchildren. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1800017553.

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.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Type VII Toxin/Antitoxin Classification System for Antitoxins that Enzymatically Neutralize Toxins.

Toxin/antitoxin (TA) systems are present in nearly all bacterial and archaeal strains and consist of a toxin that reduces growth and an antitoxin that masks toxin activity. Currently there are six primary classes for TA systems based on the nature of the antitoxin and the way that the antitoxin inactivates the toxin. Here we show that there now are at least three additional and distinct TA systems in which the antitoxin is an enzyme and the cognate toxin is the direct target of the antitoxin: Hha/TomB (antitoxin oxidizes Cys18 of the toxin), TglT/TakA (antitoxin phosphorylates Ser78 of the toxin), and HepT/MntA (antitoxin adds three AMPs to Tyr104 of the toxin). Thus, we suggest the type VII TA system should be used to designate those TA systems in which the enzyme antitoxin chemically modifies the toxin post-translationally to neutralize it. Defining the type VII TA system using this specific criterion will aid researchers in classifying newly discovered TA systems as well as refine the framework for recognizing the diverse biochemical functions in TA systems.

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.

Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens.

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3'-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.

An Application-based programme to reinforce and maintain lower salt intake (AppSalt) in schoolchildren and their families in China.

INTRODUCTION: Salt intake is very high in China, with ≈80% being added by the consumers. It is difficult to reduce salt in such settings. Our previous study (School-based Education programme to reduce Salt(School-EduSalt)) demonstrated that educating schoolchildren, who then instructed their families to reduce the amount of salt used at home, is effective in lowering salt intake in both children and adults. Our team also developed an app called 'KnowSalt', which could help individuals to estimate their salt intake and the major sources of salt in the diet. Building on School-EduSalt and KnowSalt, we propose to develop a new app (AppSalt) focusing on salt reduction through education, target setting, monitoring, evaluation, decision support and management to achieve a progressive lower salt intake for long term. To evaluate the effectiveness of the AppSalt programme, we will carry out a cluster randomised controlled trial. METHODS AND ANALYSIS: We will recruit 54 primary schools from urban and rural areas of three provinces in China. A total of 594 children aged 8-9 years and 1188 adult family members will be randomly selected for evaluation. After baseline assessment, schools will be randomly allocated to either the intervention or control group. Children in the intervention group will be taught, with support of AppSalt, about salt reduction and assigned homework to get the whole family involved in the activities to reduce salt consumption. The duration of the intervention is two school terms (ie, 1 year). The primary outcome is the difference between the intervention and control group in the change of salt intake as measured by 24-hour urinary sodium. ETHICS AND DISSEMINATION: The study has been approved by Queen Mary Research Ethics Committee and Peking University Health Science Centre IRB. Results will be disseminated through presentations, publications and social media. TRIAL REGISTRATION NUMBER: ChiCTR1800017553.

Structure and allosteric coupling of type â ¡ antitoxin CopASO.

Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the C-terminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate.

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.

Structure-function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN-MNT toxin-antitoxin system.

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite RX4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the RX4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family.

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.

Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli.

Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coliIMPORTANCEEscherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis.

Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.

Characterization of the Deep-Sea Streptomyces sp. SCSIO 02999 Derived VapC/VapB Toxin-Antitoxin System in Escherichia coli.

Toxin-antitoxin (TA) systems are small genetic elements that are ubiquitous in prokaryotes. Most studies on TA systems have focused on commensal and pathogenic bacteria; yet very few studies have focused on TAs in marine bacteria, especially those isolated from a deep sea environment. Here, we characterized a type II VapC/VapB TA system from the deep-sea derived Streptomyces sp. SCSIO 02999. The VapC (virulence-associated protein) protein belongs to the PIN (PilT N-terminal) superfamily. Overproduction of VapC strongly inhibited cell growth and resulted in a bleb-containing morphology in E. coli. The toxicity of VapC was neutralized through direct protein-protein interaction by a small protein antitoxin VapB encoded by a neighboring gene. Antitoxin VapB alone or the VapB/VapC complex negatively regulated the vapBC promoter activity. We further revealed that three conserved Asp residues in the PIN domain were essential for the toxic effect of VapC. Additionally, the VapC/VapB TA system stabilized plasmid in E. coli. Furthermore, VapC cross-activated transcription of several TA operons via a partially Lon-dependent mechanism in E. coli, and the activated toxins accumulated more preferentially than their antitoxin partners. Collectively, we identified and characterized a new deep sea TA system in the deep sea Streptomyces sp. and demonstrated that the VapC toxin in this system can cross-activate TA operons in E. coli.

Identification and characterization of a HEPN-MNT family type II toxin-antitoxin in Shewanella oneidensis.

Toxin-antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes.