Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition.

Prokaryotic Argonautes (pAgos) play a vital role in host defense by utilizing short nucleic acid guides to recognize and target complementary nucleic acids. Despite being the majority of pAgos, short pAgos have only recently received attention. Short pAgos are often associated with proteins containing an APAZ domain and a nuclease domain including DUF4365, SMEK, or HNH domain. In contrast to long pAgos that specifically cleave the target DNA, our study demonstrates that the short pAgo from Thermocrispum municipal, along with its associated DUF4365-APAZ protein, forms a heterodimeric complex. Upon RNA-guided target DNA recognition, this complex is activated to nonspecifically cleave DNA. Additionally, we found that the TmuRE-Ago complex shows a preference for 5'-OH guide RNA, specifically requires a uridine nucleotide at the 5' end of the guide RNA, and is sensitive to single-nucleotide mismatches between the guide RNA and target DNA. Based on its catalytic properties, our study has established a novel nucleic acid detection method and demonstrated its feasibility. This study not only expands our understanding of the defense mechanism employed by short pAgo systems but also suggests their potential applications in nucleic acid detection.

The mediating role of organizational commitment between workplace bullying and turnover intention among clinical nurses in China: a cross-sectional study.

BACKGROUND: Workplace bullying experienced by clinical nurses is a critical and pervasive issue that not only detrimentally impacts nurses but also poses a significant threat to the overall quality of nursing services and patient care. This study aimed to determine the mediating role of organizational commitment in the relationship between workplace bullying and turnover intention among clinical nurses in China. METHODS: Participants were recruited from 40 hospitals in various provinces of China from December 2, 2021 to February 25, 2023, using convenience sampling. After obtaining hospital ethical approval and participants' informed consent, clinical nurses (n = 585) from different nursing departments in different hospitals completed the questionnaire. The Socio-demographic Questionnaire, Negative Acts Qestionnaire, Chinese Workers' Organizational Commitment Scale and Turnover Intention Questionnaire were used to collect general demographic data of nurses and assess workplace bullying they experienced, their level of organizational commitment and turnover intention. Descriptive statistics, Pearson correlation analyses and structural equation model were adopted to analyze the data. RESULTS: Pearson's correlation analysis showed that that workplace bullying was significantly negatively correlated with organizational commitment (r = - 0.512, P<0.01) and significantly positively correlated with turnover intention (r = 0.558, P<0.01), organizational commitment was significantly negatively correlated with turnover intention (r = - 0.539, P<0.01). Mediation analysis indicated organizational commitment partially mediated the association between workplace bullying and turnover intention. The total effect (ß = 0.69) of workplace bullying on turnover intention consisted of its direct effect (ß = 0.41) and the indirect effect mediated through organizational commitment (ß = 0.280), with the mediating effect accounting for 40.58% of the total effect. CONCLUSION: Organizational commitment mediated the associations of workplace bullying and turnover intention. Therefore, healthcare organizations and nursing managers should develop appropriate strategies to enhance nurses' organizational commitment in order to reduce their turnover intention.

Behavioral Health Service Use Among Licensed Social Workers: A Qualitative Inquiry.

This paper presents qualitative data collected from 996 licensed social workers in the United States who reported mental health and/or alcohol and other drug problems and indicated the types of services they used to address these issues. Outpatient therapy was the most commonly accessed modality to treat mental health issues. Regarding problems with alcohol and other drugs, self-help groups were the most frequently utilized intervention. Qualitative findings suggest that behavioral health service use has influenced respondents' work with clients, personal and professional development, and career trajectories. Barriers to service use, such as stigma and limited access to care, were also identified. Implications for social work education and professional practice are discussed.

Prokaryotic Gabija complex senses and executes nucleotide depletion and DNA cleavage for antiviral defense.

The Gabija complex is a prokaryotic antiviral system consisting of the GajA and GajB proteins. GajA was identified as a DNA nicking endonuclease but the functions of GajB and the complex remain unknown. Here, we show that synergy between GajA-mediated DNA cleavage and nucleotide hydrolysis by GajB initiates efficient abortive infection defense against virulent bacteriophages. The antiviral activity of GajA requires GajB, which senses DNA termini produced by GajA to hydrolyze (d)A/(d)GTP, depleting essential nucleotides. This ATPase activity of Gabija complex is only activated upon DNA binding. GajA binds to GajB to form stable complexes in vivo and in vitro. However, a functional Gabija complex requires a molecular ratio between GajB and GajA below 1:1, indicating stoichiometric regulation of the DNA/nucleotide processing complex. Thus, the Gabija system exhibits distinct and efficient antiviral defense through sequential sensing and activation of nucleotide depletion and DNA cleavage, causing a cascade suicide effect.

A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus.

Prokaryotes possess numerous diverse defense systems to resist viral infections, while some viruses have also evolved antiviral defense systems to exclude other viruses in cases of multiple infections. Here, we report the first virus-derived modification-dependent restriction endonuclease (HHPV4I) from the archaeal virus HHPV4 (Haloarcula hispanica pleomorphic virus 4). HHPV4I contains an SRA domain, a winged helix (wH) domain, and an HNH domain; recognizes the Gm6ATC site; and specifically binds to Gm6ATC site-containing DNA. Both the wH domain and the HNH domain are responsible for DNA binding. Unlike the well-known m6A-specific restriction enzyme DpnI, HHPV4I only efficiently cleaves DNA with a fully methylated Gm6ATC site and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. Furthermore, HHPV4I preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC site. Thus, the cleavage pattern of HHPV4I is distinct from those of all of the presently characterized restriction endonucleases. Mutations in the wH domain of HHPV4I do not alter m6A-dependent endonuclease activity, but they decrease recognition sequence specificity, thus expanding the cleaving capacity to more m6A-containing DNA sequences. The wH domain provides a target for searching, developing, and engineering novel m6A-dependent endonucleases. IMPORTANCE Many modification-dependent restriction endonucleases (MDREs) were identified in prokaryotes and recognized modified cytosine bases, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and glucosyl-5-hydroxymethylcytosine (g5hmC). The first virus-derived MDRE (HHPV4I) from the archaeal virus HHPV4 was identified in this study. The viral MDRE suggested a new strategy employed by the virus to exclude other viruses in the case of multiple replications. HHPV4I is a novel N6-methyladenine (m6A)-dependent restriction endonuclease, while the cleavage pattern of HHPV4I is distinct from the well-known m6A-dependent restriction endonuclease DpnI. HHPV4I recognizes Gm6ATC sites and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. It preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC sites. Furthermore, mutations in the HHPV4I wH domain can alter the sequence specificity without impeding the m6A-dependent DNA cleavage activity, providing a target for engineering more m6A-dependent endonucleases with different sequence specificities.

Design, synthesis and neurite outgrowth activity of novel ganglioside GM1 derivatives by remodeling of the fatty acid moiety.

Ganglioside GM1 is a glycosphingolipid found on mammalian cell membranes, and it is involved in ischemic encephalopathy, spinal cord injury and neurodegenerative diseases. Fatty acids, as a structure module of GM1, have been reported to affect its physiological function and neurite growth activity. Due to the limitation of preparation methods, the function of GM1 derivatives containing different fatty acids in nerve cells has not been systematically studied. To discover novel GM1 derivatives as nerve growth-promoting agents, we developed an efficient SA_SCDase enzymatic synthetic system of GM1 derivatives, yielding twenty GM1 derivatives with unsaturated fatty acid chains in high total yields (16-67%). Subsequently, the neurite outgrowth activities of GM1 derivatives were assessed on Neuro2a Cells. Among all the GM1 derivatives, GM1 (d18:1/C16:1) induced demonstrably neurite outgrowth activity. The subsequent RNA-sequencing (RNA-seq) and Western blot analysis was then performed and indicated that the mechanism of nerve cells growth involved cholesterol biosynthesis regulation by up-regulating SREBP2 expression or ERBB4 phosphorylation to activate the PI3K-mTOR pathway.

A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase.

Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

A nucleotide-sensing endonuclease from the Gabija bacterial defense system.

The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.

Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase.

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

Corrigendum: Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase.

[This corrects the article DOI: 10.3389/fmicb.2021.766612.].

The Cyclic Oligoadenylate Signaling Pathway of Type III CRISPR-Cas Systems.

Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently.

Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis.

We have characterized the single subunit RNA polymerase from Klebsiella phage KP34. The enzyme is unique among known bacteriophage RNA polymerases in that it recognizes two unrelated promoter sequences, which provided clues for the evolution of phage single-subunit RNA polymerases. As the first representative enzyme from the "phiKMV-like viruses" cluster, its use in run-off RNA synthesis was investigated. RNA-Seq analysis revealed that the KP34 RNA polymerase does not possess the undesired self-templated RNA terminus extension known for T7 RNA polymerase and is suitable to synthesize RNAs with structured 3' termini such as sgRNAs. A KP34 RNA polymerase Y603F mutant is engineered to incorporate deoxy- and 2'-fluoro ribonucleotide into RNA.

A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production.

Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca(2+) and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.

Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids.

Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.

Generation and characterization of monoclonal antibodies against channel catfish virus.

Three monoclonal antibodies (MAbs) against channel catfish virus (CCV) were generated from mice immunized with purified CCV. Western blot analysis revealed that the MAb 3G12 reacted with three CCV proteins of 94 kDa, 130 kDa, and 170 kDa; the MAb 4C4 reacted with two CCV proteins of 130 kDa and 170 kDa; and the MAb 4D4 reacted with two CCV proteins of 94 kDa and 98 kDa. Indirect immunofluorescence assay showed intense fluorescence in the CCV-infected channel catfish ovary (CCO) cells in areas corresponding to the location of granular structures. In addition, the three MAbs could completely neutralize CCV at a dilution of 1:500. This study demonstrated that these MAbs could recognize CCV specifically and will be useful in the development of diagnostic methods for the detection of fish CCV infection.

Efficient gene delivery into fish cells by an improved recombinant baculovirus.

Studies on transduction of mammalian cells have shown that baculovirus can be used as an effective vector for gene delivery. However, previous studies have found the gene delivery efficiency to be very low in differentiated fish cells. In this study, an improved recombinant baculovirus, containing cytomegalovirus immediate-early (CMV-IE) promoter and an enhanced green fluorescent protein (EGFP) gene as the reporter gene, was constructed. The transduction efficiency of recombinant baculovirus in several fish cell lines was measured by flow cytometry (FCM) and the persistence of EGFP was monitored by fluorescence microscopy. The results demonstrated that baculovirus can mediate the high efficiency of gene delivery into differentiated fish cells. Furthermore, it was found that growth medium is superior to PBS as the surrounding solution to enhance the transduction efficiency in some fish cells. In addition, transgene expression can persist for a lengthy period in fish cells, and attained highest level several days later after transduction. This study suggest that the improved recombinant baculovirus can be an excellent gene delivery vector for fish cells and also providing new insights into the transduction of vertebrate cells with baculovirus.