Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories.

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis.

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

Immunodiversity of the Arabidopsis ZAR1 NLR Is Conveyed by Receptor-Like Cytoplasmic Kinase Sensors.

The Arabidopsis nucleotide-binding leucine-rich repeat protein ZAR1 can recognize at least six distinct families of pathogenic effector proteins to mount an effector-triggered immune response. This remarkable immunodiversity appears to be conveyed by receptor-like cytoplasmic kinase (RLCK) complexes, which associate with ZAR1 to sense several effector-induced kinase perturbations. Here we show that the recently identified ZAR1-mediated immune responses against the HopX1, HopO1, and HopBA1 effector families of Pseudomonas syringae rely on an expanded diversity of RLCK sensors. We show that individual sensors can recognize distinct effector families, thereby contributing to the expanded surveillance potential of ZAR1 and supporting its role as a guardian of the plant kinome.

Perturbations of the ZED1 pseudokinase activate plant immunity.

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.

Identifying Pseudomonas syringae Type III Secreted Effector Function via a Yeast Genomic Screen.

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e., congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.

Mycobacterium tuberculosis origin of replication and the promoter for immunodominant secreted antigen 85B are the targets of MtrA, the essential response regulator.

Efficient proliferation of Mycobacterium tuberculosis (Mtb) inside macrophage requires that the essential response regulator MtrA be optimally phosphorylated. However, the genomic targets of MtrA have not been identified. We show by chromatin immunoprecipitation and DNase I footprinting that the chromosomal origin of replication, oriC, and the promoter for the major secreted immunodominant antigen Ag85B, encoded by fbpB, are MtrA targets. DNase I footprinting analysis revealed that MtrA recognizes two direct repeats of GTCACAgcg-like sequences and that MtrA approximately P, the phosphorylated form of MtrA, binds preferentially to these targets. The oriC contains several MtrA motifs, and replacement of all motifs or of a single select motif with TATATA compromises the ability of oriC plasmids to maintain stable autonomous replication in wild type and MtrA-overproducing strains, indicating that the integrity of the MtrA motif is necessary for oriC replication. The expression of the fbpB gene is found to be down-regulated in Mtb cells upon infection when these cells overproduce wild type MtrA but not when they overproduce a nonphosphorylated MtrA, indicating that MtrA approximately P regulates fbpB expression. We propose that MtrA is a regulator of oriC replication and that the ability of MtrA to affect apparently unrelated targets, i.e. oriC and fbpB, reflects its main role as a coordinator between the proliferative and pathogenic functions of Mtb.

CtrA, a global response regulator, uses a distinct second category of weak DNA binding sites for cell cycle transcription control in Caulobacter crescentus.

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA approximately P exhibits high affinity (dissociation constant [K(d)], <10 nM) for consensus TTAA-N7-TTAA binding sites with "typical" (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (K(d), >500 nM) CtrA binding sites with "atypical" (N not equal 7) spacing. Footprints demonstrated that phosphorylated CtrA approximately P does not exhibit increased affinity for "atypical" sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 microM) accumulate, which can drive CtrA binding to "atypical" sites. In vivo cross-linking showed that when the stable CtrADelta3 protein persists during the cell cycle, the "atypical" sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrADelta3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA approximately P phosphorylation that provides the dominant "activation" signal. Protein dimerization is one potential means of "activation." The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the "atypical" sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how "atypical" sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

CtrA response regulator binding to the Caulobacter chromosome replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression.

The Caulobacter crescentus chromosome replication origin (Cori) has five binding sites for CtrA, an OmpR/PhoB family 'response regulator'. CtrA is degraded in replicating 'stalked' cells but is abundant in the non-replicating 'swarmer' cells, where it was proposed to repress replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid replication and in the natural chromosome locus. Remarkably, the C. crescentus chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled chromosome replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA.