Validation of species-specific PCR assays for the detection of Pantoea ananatis, P. agglomerans, P. allii, and P. stewartii

Species of Pantoea represent a group of plant pathogenic bacteria that infect a variety of agro-economically important plant species. Among these, a complex of P. ananatis, P. allii, P. agglomerans, and P. stewartii subsp. indologenes cause center rot in onion, resulting in significant economic losses. As species of Pantoea are phenotypically closely related, identification of Pantoea species relies on the sequencing and phylogenetic analysis of housekeeping genes. To aid in rapid identification of Pantoea species, efforts have been made in developing species-specific primers to be used in PCR assays. In the current study, two P. ananatis, one P. allii, one P. agglomerans, and three P. stewartii published primers as well as newly developed P. agglomerans PagR primers were evaluated for their specificity against 79 Pantoea strains, belonging to 15 different species. To ensure that selected primers were evaluated against accurately identified species, sequencing and phylogenetic analysis of housekeeping gene infB were conducted. Thereafter, PCR assays using selected species-specific primers were performed. The results showed that previously described P. ananatis-specific PANA_1008; P. allii-specific allii-leuS; P. stewartii-specific PANST_rpoB, 3614galE, and DC283galE primers; and one newly designed P. agglomerans-specific PagR primer pair were highly specific for their target Pantoea species. They accurately identified these strains into their species and, in some cases, their subspecies level. The findings of the current study will facilitate rapid and reliable identification of P. ananatis, P. agglomerans, P. allii, and P. stewartii.USDA, National Institute of Food and Agriculture.https://apsjournals.apsnet.org/loi/pdishj2023BiochemistryGeneticsMicrobiology and Plant Patholog

Draft genome sequences for Pantoea ananatis ATCC 35400 and Pantoea stewartii subspecies indologenes ICMP 10132

DATA AVAILABILITY : This Whole Genome Shotgun project for P. ananatis ATCC 35400 has been deposited in DDBJ/ENA/GenBank under accession no. JARNMU000000000. The version described in this paper is the first version, JARNMU010000000. Raw reads used to generate this assembly can be found at SRX19856715, SRX19856716, and SRX19856717. This Whole Genome Shotgun project for P. stewartii ICMP 10132 has been deposited in DDBJ/ENA/ GenBank under accession no. JARNMT000000000. The version described in this paper is the first version, JARNMT010000000. Raw reads used to generate this assembly can be found at SRX19854976 and SRX19854977.Here, we describe draft genome sequences for two bacterial isolates from the genus Pantoea. Pantoea ananatis ATCC 35400 was originally isolated from honeydew melon and was obtained from the American Type Culture Collection. Pantoea stewartii subspecies indologenes ICMP 10132 was originally isolated from sugarcane and classified as Pantoea ananatis, but average nucleotide identity and discriminatory PCR support species reclassification.The National Science Foundation (NSF).https://journals.asm.org/journal/mrahj2024BiochemistryGeneticsMicrobiology and Plant PathologySDG-15:Life on lan

In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model

Bioluminescent imaging (BLI) technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real-time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5 × 103 bacteria and monitored by BLI at 24, 48, and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant antieffector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000

A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

<p>Abstract</p> <p>Background</p> <p>The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (<it>ori</it>_R6Kγ) is dependent on the <it>pir </it>gene-encoded π protein. Originally encoded by R6K, this protein is usually provided <it>in trans </it>in hosts engineered to support replication of plasmids harboring <it>ori</it>_R6Kγ. In <it>Escherichia coli </it>this is commonly achieved by chromosomal integration of <it>pir </it>either via lysogenization with a λ<it>pir </it>phage or homologous recombination at a pre-determined locus.</p> <p>Findings</p> <p>Current methods for construction of host strains for <it>ori</it>_R6Kγ-containing plasmids involve procedures that do not allow selection for presence of the <it>pir </it>gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn<it>7</it>-based method for rapid and reliable construction of <it>pir</it>⁺host strains. Using a curable mini-Tn<it>7 </it>delivery plasmid, <it>pir </it>expressing derivatives of several commonly used <it>E. coli </it>cloning and mobilizer strains were isolated using both the wild-type <it>pir⁺</it>gene as well as the copy-up <it>pir-116 </it>allele. In addition, we isolated <it>pir</it>⁺and <it>pir-116 </it>expressing derivatives of a clinical isolate of <it>Salmonella enterica </it>serovar Typhimurium. In both <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium, the presence of the <it>pir⁺</it>wild-type or <it>pir-116 </it>alleles allowed the replication of <it>ori</it>_R6Kγ-containing plasmids.</p> <p>Conclusions</p> <p>A mini-Tn<it>7 </it>system was employed for rapid and reliable engineering of <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium host strains for plasmids containing <it>ori</it>_R6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with <it>ori</it>_R6Kγ.</p

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant antieffector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000

Pantailocins : phage-derived bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes

SUPPLEMENTARY MATERIAL : FILE S1. Alignment file for sheath protein sequences to recreate phylogenies in manuscript. FILE S2. Alignment file for J plate protein sequences to recreate phylogenies in manuscript. FIGURE S1. A Model For Invertase Activity in the P. stewartii ICMP 10132 Tailocin Locus.ERRATUM : Volume 89, no. 12, e00471-23, 2023, https://doi.org/10.1128/aem.00929-23. The affiliations and the affiliation numbers for each author should appear as shown in this erratum.Phage-derived bacteriocins are highly specific and effective antimicrobial molecules, which have successfully been used as prophylactic treatments to prevent phytopathogen infections. Given the specificity of tailocins, a necessary step for broadening the tailocin catalog and for extending applicability across systems and diseases is the screening of new clades of phytopathogens for the production of molecules with tailocin-like killing activity. Here, we describe the production by and sensitivity of strains to tailocins produced by Pantoea ananatis and Pantoea stewartii subsp. indologenes. Phylogenetic evidence suggests that these tailocins are derived from Myoviridae family phage like many previously described R-type tailocins but also suggests that cooption from phage occurred independently of previously described tailocins. Since these tailocin encoding loci are present in the same genomic locations across multiple strains of both species and display a level of divergence that is consistent with other shared regions between the genomes and with vertical inheritance of the locus, we refer to them broadly as “Pantailocins.” IMPORTANCE : Phage-derived bacteriocins (tailocins) are ribosomally synthesized structures produced by bacteria in order to provide advantages against competing strains under natural conditions. Tailocins are highly specific in their target range and have proven to be effective for the prevention and/or treatment of bacterial diseases under clinical and agricultural settings. We describe the discovery and characterization of a new tailocin locus encoded within genomes of Pantoea ananatis and Pantoea stewartii subsp. indologenes, which may enable the development of tailocins as preventative treatments against phytopathogenic infection by these species.National Science Foundation (NSF).https://journals.asm.org/journal/aemhj2024BiochemistryGeneticsMicrobiology and Plant PathologySDG-15:Life on lan

The distribution of onion virulence gene clusters among Pantoea spp.

Pantoea ananatis is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and alt, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of pantaphos, (2-hydroxy[phosphonomethyl) maleate), a phosphonate phytotoxin. The alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggests that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea spp. were phenotyped using the red onion scale necrosis (RSN) assay and were genotyped using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946–2019, was tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (P < 0.001). There is also a significant association of a positive HiVir PCR and RSN assay among P. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN C /HiVir C /alt C ] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN C /HiVir C /alt] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.The Vidalia Onion Committee, United States Department of Agriculture (USDA), ANII, Uruguay), CSIC Grupos de Investigación I + D 2000 (CSIC, Udelar, Uruguay), Specialty Crops Research Initiative Award from the USDA, and National Institute of Food and Agriculture.http://www.frontiersin.org/Plant_Scienceam2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Genomic delineation and description of species and within-species lineages in the genus Pantoea

As the name of the genus Pantoea (“of all sorts and sources”) suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content dierences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified

Genomic delineation and description of species and within-species lineages in the genus Pantoea

DATA AVAILABILITY STATEMENT : The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, PRJNA445714.As the name of the genus Pantoea (“of all sorts and sources”) suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.The National Science Foundation and the United States Department of Agriculture—National Institute of Food and Agriculture. In part by the Virginia Agricultural Experiment Station, the Hatch Program of USDA NIFA and the National Institute of Health.http://www.frontiersin.org/Microbiologyam2024BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologySDG-15:Life on lan