Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Gram-negative endolysins: overcoming the outer membrane obstacle.

Our ability to control the growth of Gram-negative bacterial pathogens is challenged by rising antimicrobial resistance and requires new approaches. Endolysins are phage-derived enzymes that degrade peptidoglycan and therefore offer potential as antimicrobial agents. However, the outer membrane (OM) of Gram-negative bacteria impedes the access of externally applied endolysins to peptidoglycan. This review highlights recent advances in the discovery and characterization of natural endolysins that can breach the OM, as well as chemical and engineering approaches that increase antimicrobial efficacy of endolysins against Gram-negative pathogens.

Genome sequences of Mx1, the first Myxococcus phage isolated, and Mx4, a generalized transducing myxophage.

Myxococcus xanthus is the best-studied member of the phylum Myxococcota, but the bacteriophages infecting it and their characterization remain limited. Here, we present complete genomes of Mx1, the first Myxococcus phage isolated, and of an Mx4 derivative widely used for generalized transduction, both unclassified Caudoviricetes with long, contractile tails.

Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection.

Bacteria have diverse defenses against phages. In response, jumbo phages evade multiple DNA-targeting defenses by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognizing viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type-III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types.

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.

PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.

A mobile restriction-modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction-modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

SorTn-seq: a high-throughput functional genomics approach to discovering regulators of bacterial gene expression.

We recently developed a high-throughput functional genomics approach, named 'SorTn-seq', to identify factors affecting expression of any gene of interest in bacteria. Our approach facilitates high-throughput screening of complex mutant pools, a task previously hindered by a lack of suitable techniques. SorTn-seq combines high-density, Tn5-like transposon mutagenesis with fluorescence-activated cell sorting of a strain harboring a promoter-fluorescent reporter fusion, to isolate mutants with altered gene expression. The transposon mutant pool is sorted into different bins on the basis of fluorescence, and mutants are deep-sequenced to identify transposon insertions. DNA is prepared for sequencing by using commercial kits augmented with custom primers, enhancing ease of use and reproducibility. Putative regulators are identified by comparing the number of insertions per genomic feature in the different sort bins, by using existing bioinformatic pipelines and software packages. SorTn-seq can be completed in 1-2 weeks and requires general microbiology skills and basic flow cytometry experience.

The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR-Cas immune systems.

CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR-Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR-Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR-Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.

Crystal structure of the anti-CRISPR repressor Aca2.

Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.

Evolution of virulence in a novel family of transmissible mega-plasmids.

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; amber disease-associated plasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.

The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.

Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.

Applying Evidence-Centered Design to Measure Psychological Resilience: The Development and Preliminary Validation of a Novel Simulation-Based Assessment Methodology.

Modern technologies have enabled the development of dynamic game- and simulation-based assessments to measure psychological constructs. This has highlighted their potential for supplementing other assessment modalities, such as self-report. This study describes the development, design, and preliminary validation of a simulation-based assessment methodology to measure psychological resilience-an important construct for multiple life domains. The design was guided by theories of resilience, and principles of evidence-centered design and stealth assessment. The system analyzed log files from a simulated task to derive individual trajectories in response to stressors. Using slope analyses, these trajectories were indicative of four types of responses to stressors: thriving, recovery, surviving, and succumbing. Using Machine Learning, the trajectories were predictive of self-reported resilience (Connor-Davidson Resilience Scale) with high accuracy, supporting construct validity of the simulation-based assessment. These findings add to the growing evidence supporting the utility of gamified assessment of psychological constructs. Importantly, these findings address theoretical debates about the construct of resilience, adding to its theory, supporting the combination of the "trait" and "process" approaches to its operationalization.

Complete Genome Sequences of the Escherichia coli Donor Strains ST18 and MFDpir.

Escherichia coli ST18 and MFDpir are donors commonly used to transfer oriT RP4-containing plasmids to diverse bacteria via conjugation. ST18 and MFDpir were constructed via multiple genetic manipulations involving several E. coli strains. Here, we used Illumina and Nanopore sequencing to determine the complete genomes of these widely used strains.

Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia.

Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin-antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to 'shut down', limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.

A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.

CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages1. However, DNA modification2,3, the production of anti-CRISPR proteins4,5 and potentially other strategies enable phages to evade CRISPR-Cas. Here, we discovered a Serratia jumbo phage that evades type I CRISPR-Cas systems, but is sensitive to type III immunity. Jumbo phage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shell-a phenomenon only reported recently for distantly related Pseudomonas phages6,7. All three native CRISPR-Cas complexes in Serratia-type I-E, I-F and III-A-were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbo phage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbo phages that were identified in global viral sequence datasets. The ability to recognize jumbo phage RNA and elicit immunity probably contributes to the presence of both RNA- and DNA-targeting CRISPR-Cas systems in many bacteria1,8. Together, our results support the model that jumbo phage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting, defences, and that this phenomenon is widespread among jumbo phages.