Bacterial Cell Lysate Research Articles

Humanized recombinant immunotoxin targeting hCG demonstrates therapeutic potential for advanced stage difficult to treat cancers

We report the development of an immunotherapeutic molecule, a Humanized immunotoxin, for treating hCG-expressing advanced-stage cancers. PiPP, a high-affinity anti-hCG monoclonal antibody, is used in the immunotoxin for ‘homing’ hCG-positive cancer cells. The deimmunized (DI) form of α-Sarcin, a fungal-origin toxin that lacks functional T-cell epitopes, is used in the design to ensure minimal immunogenicity of the immunotoxin for repetitive use in humans. A single-chain variable fragment (scFv) of PiPP was constructed by linking the Humanized VH and VL regions of the antibody. The scFv part of the antibody was further linked to the toxin α-Sarcin (DI) at the gene level and expressed as a recombinant protein in E. coli. The immunotoxin was purified from the bacterial cell lysate and analysed for binding and cytotoxicity to hCG-secreting colorectal and pancreatic cancer cells. The results showed that the scFv(PiPP)-Sarcin immunotoxin was able to bind to colorectal and pancreatic cancer cells and killed approximately 85% of the cells. In vivo testing of the immunotoxin produced results similar to those of in vitro testing against colorectal adenocarcinoma-induced tumours. This immunotoxin could be a promising immunotherapeutic agent for treating colorectal, pancreatic and other terminal-stage hCG-expressing cancers.

Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9 % lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5 %) and purity (98.1 %) were obtained with 1,3-dioxolane at 80°C and 6 h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor culture leading to the production of 78.7 gPHB/L, 56.9 % (w/w) PHB content and 2.3 g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.

Bacterial Cell Lysates of Bacillus sp. F Isolated From Permafrost Diminish DNA Damage by Hydrogen Peroxide in Murine Blood Cells Ex Vivo

Nowadays, there is growing interest in new compositions and drugs that can preserve and maintain human health. There are many studies on both known probiotics and new sources of biologically active substances which may potentially be new avenues for human longevity. Here, we describe the protective effect of cell lysates of the relict bacteria Bacillus sp. F (Bacilus Lyuba), isolated from the ancient permafrost of Central Yakutia, upon DNA damage, induced by hydrogen peroxide. The DNA damage was monitored by a comet assay in leukocytes isolated from the peripheral blood of experimental animals (BALB/c mice). Cell lysates were prepared using the French Press and ultrasonic treatment. The levels of DNA damage in the presence of bacterial lysates upon the stress test (20 μM hydrogen peroxide for 10 min at 37°C) were almost twice lower, compared with the buffer control. Of note, the maximal protective effect was observed in the temperature range of 50°C–60°C; with the increase of the temperature, this effect vanished. The obtained results do not give a clear answer on the mechanism of the protective properties of Bacillus sp. F. (Bacilus Lyuba) lysates. To answer this question, a detailed analysis of the protein composition of bacterial lysate at different temperatures should be performed.

Quantitative detection of formaldehyde using solid phase microextraction gas chromatography–mass spectrometry coupled to cysteamine scavenging

Formaldehyde (HCHO) is a toxic and carcinogenic pollutant and human metabolite that reacts with biomolecules under physiological conditions. Quantifying HCHO is essential for ongoing biological and biomedical research on HCHO; however, its reactivity, small size and volatility make this challenging. Here, we report a novel HCHO detection/quantification method that couples cysteamine-mediated HCHO scavenging with SPME GC–MS analysis. Our NMR studies confirm cysteamine as an efficient and selective HCHO scavenger that out-competes O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, the most commonly used scavenger, and forms a stable thiazolidine amenable to GC–MS quantification. Validation of our GC–MS method using FDA and EMA guidelines revealed detection and quantification limits in the nanomolar and micromolar ranges respectively, while analysis of bacterial cell lysate confirmed its applicability in biological samples. Overall, our studies confirm that cysteamine scavenging coupled to SPME GC–MS analysis provides a sensitive and chemically robust method to quantify HCHO in biological samples.

Exploring the impact of nucleic acids on protein stability in bacterial cell lysate

- Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion between protein molecules.- However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within the lysate could enhance protein stability via electrostatic interactions.- Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation activity toward proteins involving electrostatic interactions.- Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or medium commonly used in experiments.- The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays using cell lysate or samples containing nucleic acids.

Genomic DNA causes membrane fouling during sterile filtration of cell lysates

During biopharmaceutical production, efficient removal of impurities via filtration is essential. It is desirable to achieve high filter capacities along with a high degree of clearance. However, the complexity of bacterial cell lysates makes it difficult to properly estimate the filtration behavior. Here we investigated how host cell-derived impurities impacted the performance of commercially available sterile filters, with a focus on characterizing the dsDNA-induced decrease of filter capacity. Pressure flow curves at a constant flow, combined with particle analytics (e.g., nanoparticle-tracking analysis), indirectly revealed that dsDNA had a major influence on rapid membrane fouling. The observed phenomenon was more pronounced when using filters with small pore sizes (<0.2 µm). Filter capacity could be substantially increased by reducing the size of the present dsDNA fragments, or by decreasing the potential for dsDNA-driven electrostatic interactions with positively charged molecules. In general, our present findings highlight the importance of monitoring dsDNA even during the primary recovery of proteins in cell lysates.

Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

Diffusion NMR and Rheology of a Model Polymer in Bacterial Cell Lysate Crowders.

The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (Rg ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins

BackgroundIn spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host’s capsule.ResultsThe TP84_26 ‘capsule depolymerase’ (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC–MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion.ConclusionsThe novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Top-down proteomic identification of protein biomarkers of Xylella fastidiosa subsp. fastidiosa using MALDI-TOF-TOF-MS and MS/MS

Protein biomarkers were identified from bacterial cell lysates of two sequenced strains of Xylella fastidiosa subsp. fastidiosa (Xff), the causal agent of Pierce's Disease of grapevine, using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. Proteins were identified from their intact masses as well as sequence-specific fragment ions resulting from the aspartic acid effect detected by MS/MS and post-source decay (PSD). Two Xff strains from California were studied, M23 isolated from almond in Kern County and Stag's Leap isolated from grapevine in Napa County. The two strains are pathogenically identical or highly similar. Most of the proteins identified were highly conserved, e.g., 10 kDa chaperonin, cold-shock and/or DNA-binding, ribosomal and hypothetical proteins. The amino acid sequences and post translational modifications (PTM) of these conserved proteins were identical between the two Xff strains. However, the amino acid sequence of an outer membrane/hypothetical protein differed significantly between the two strains and could be a useful biomarker to distinguish between these closely related strains. In silico signal peptide analysis suggest that the outer membrane/hypothetical protein are secreted lipoproteins (SPII) with a putative prosthetic group attached to the only cysteine residue (C19) before or after removal of an 18-residue N-terminal signal peptide. However, only y-type fragment ions were detected by MS/MS-PSD which precluded confirmation of the site of sequence truncation and/or attached PTM. The annotated genomes of the two strains indicate ten to twenty proteins that are either lipoproteins or are associated with assembly, modification, or transport of lipoproteins. In consequence, a variety of prosthetic groups, e.g. diacylglycerol, may be attached to C19 in the mature protein.

The enzyme activity of sortase A is regulated by phosphorylation in Staphylococcus aureus

ABSTRACT In many Gram-positive bacteria, the transpeptidase enzyme sortase A (SrtA) anchors surface proteins to cell wall and plays a critical role in the bacterial pathogenesis. Here, we show that in Staphylococcus aureus, an important human pathogen, the SrtA is phosphorylated by serine/threonine protein kinase Stk1. S. aureus SrtA can also be phosphorylated by small-molecule phosphodonor acetyl phosphate (AcP) in vitro. We determined that various amino acid residues of S. aureus SrtA are subject to phosphorylation, primarily on its catalytic site residue cysteine-184 in the context of a bacterial cell lysate. Both Stk1 and AcP-mediated phosphorylation inhibited the enzyme activity of SrtA in vitro. Consequently, deletion of gene (i.e. stp1) encoding serine/threonine phosphatase Stp1, the corresponding phosphatase of Stk1, caused an increase in the phosphorylation level of SrtA. The stp1 deletion mutant mimicked the phenotypic traits of srtA deletion mutant (i.e. attenuated growth where either haemoglobin or haem as a sole iron source and reduced liver infections in a mouse model of systemic infection). Importantly, the phenotypic defects of the stp1 deletion mutant can be alleviated by overexpressing srtA. Taken together, our finding suggests that phosphorylation plays an important role in modulating the activity of SrtA in S. aureus.

Effects of Postbiotic from Bacteriocin-Like Inhibitory Substance Producing Enterococcus faecalis on Toxigenic Clostridioides difficile

Objective: To determine the activities of postbiotics, prepared from bacteriocin-like producing Enterococcus faecalis (E. faecalis) against Clostridioides difficile (C. difficile) and its spores, and to assess the safety of postbiotics in vivo. Material and Methods: Bacteriocin production of E. faecalis PK1201 was screened by proteolytic enzyme treatment, and bacteriocin-encoding genes were characterized by whole genome sequencing. A postbiotic of E. faecalis PK1201 was prepared using neutralized cell-free supernatant or bacterial cell lysate, which was then used to screen for antimicrobial activity via agar well diffusion. The lyophilized cell-free supernatant (LCFS) was further determined for its minimum inhibitory concentration (MIC) against C. difficile 630. The morphological changes of C. difficile were observed under scanning electron microscopes (SEM). Subsequently, the LCFS at sub-MIC and MIC were used to evaluate anti-spore germination activity. Finally, the safety of postbiotics was accessed using the Galleria mellonella model. Results: E. faecalis PK1201 carried enterolysin A encoding gene. For postbiotic preparation, only LCFS exhibited antimicrobial activity, and the activity was completely lost after proteinase K treatment; indicating the existence of bacteriocin. LCFS showed anti-C. difficile with MIC of 18.2±6.9 mg/mL. The SEM images demonstrated shorter destruction of C. difficile cells after being treated with LCFS. Interestingly, LCFS at the MIC and sub-MIC revealed anti-spore germination activity against toxigenic C. difficile compared to the control. LCFS showed no acute toxicity in G. mellonella at the tested concentration. Conclusion: The powerful activity and safety of LCFS shed light on the role of postbiotics in pharmaceutical products to control CDI.

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at ∼18 and ∼22 µg/mL of rHALT-1 purified with phosphate and acetate buffers, respectively.•HALT-1 is a soluble α-pore-forming toxin of 18.38 kDa.•rHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography.•The cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported.

A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices.

Phosphite, the anion of phosphorus acid, is an important metabolite in the global biogeochemical phosphorus cycle and a phosphorus species with unique agricultural properties. As such, methods for detecting phosphite quantitatively and selectively are critical to evidencing phosphorus redox chemistry. Here, we present a fluorescence-based assay for phosphite, utilizing the NAD+-dependent oxidation of phosphite by phosphite dehydrogenase and the subsequent reduction of resazurin to resorufin. With the application of a thermostable phosphite dehydrogenase, a medium-invariant analytical approach, and novel sample preparation methods, the assay is capable of rapid and accurate phosphite quantification with a 3 μM limit of detection in a wide array of biologically- and environmentally-relevant matrices, including bacterial and archaeal cell lysate, seawater, anaerobic digester sludge, and plant tissue. We demonstrate the utility of the assay by quantitating phosphite uptake in a model crop plant in the presence or absence of a phosphite-oxidising strain of Pseudomonas stutzeri as a soil additive, establishing this bacterium as an efficient phosphite converting biofertilizer.

Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA.

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

Diffusion NMR of alpha-synuclein in the presence of bacterial cell lysate crowders

The crowded and heterogeneous intracellular milieu has a concentration of macromolecules up to 400 mg/ml. The causes of macromolecular crowding effects are excluded volume and ‘soft' nonspecific interactions, such as hydrogen bonds, electrostatic, Van der Waals, and hydrophobic interactions. Some physiological consequences of macromolecular crowding on proteins are on protein structure, protein folding, phase separation, aggregation, and protein-protein interaction. Traditionally, macromolecular crowding effects has been studied in artificial crowders like Ficoll and Dextran.

Robotic Affinity Purification of Soluble and Insoluble Recombinant Glutathione-S-Transferase Fusion Proteins.

A completely automated purification of glutathione-S-transferase (GST) fusion proteins, either in soluble form or after renaturation of insoluble inclusion bodies, is described. Depending on the expression levels and the amount of glutathione affinity matrix employed, the protocol yields approximately 30-100μg of purified GST-fusion protein from 2mL microplate cultures. The high yield is facilitated by employing an efficient chemical/enzymatic lysis procedure for preparing bacterial cell lysates. Insoluble GST-fusion proteins are automatically refolded by a high-throughput robotic microdialysis procedure that also assesses the degree of successful refolding by integrated GST enzymatic assays and quantitation of soluble protein successfully recovered after affinity purification. For soluble GST-fusion proteins the purification procedure is normally completed within 60min, whereas urea-based denaturation-renaturation strategies typically require an additional 18h. The integration of quantitation of cell growth and affinity-purified GST-fusion protein yield allows direct comparisons of different expression constructs and the yield of soluble GST-fusion proteins to be optimized in a systematic manner.

Versatile separation of nucleotides from bacterial cell lysates using strong anion exchange chromatography

Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136M-1s-1) than PYCR1 (13.7M-1s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.