Comparative Genomic and Functional Analyses for Insights into Pantoea agglomerans Strains Adaptability in Diverse Ecological Niches.

Pantoea agglomerans inhabit diverse ecological niches, ranging from epiphytes and endophytes in plants, body of animals, and occasionally in the human system. This multifaceted bacterium contributes substantially to plant growth promotion, stress resilience, and biocontrol but can also act as a pathogen to its host. The genetic determinants underlying these diverse functions remain largely unfathomed and to uncover this phenomenon, nineteen strains of Pantoea agglomerans were selected and analyzed. Genome-to-Genome Distance Calculator (GGDC) which uses the Genome Blast Distance Phylogeny (GBDP) technique to calculate digital DDH values. Phylogenetic analysis via Genome-to-Genome distance, Average Nucleotide Identity, and Amino Acid Identity calculation revealed that all strains belonged to the genus Pantoea. However, strain 33.1 had a lower value than the threshold for the same species delineation. Bacterial Pan Genome Analysis (BPGA) Pipeline and MinPath analysis revealed genetic traits associated with environmental resilience, such as oxidative stress, UV radiation, temperature extremes, and metabolism of distinct host-specific carbohydrates. Protein-protein interactome analysis illustrated osmotic stress proteins closely linked with core proteins, while heavy metal tolerance, nitrogen metabolism, and Type III and VI secretion systems proteins generally associated with pathogenicity formed a separate network, indicating strain-specific characteristics. These findings shed new light on the intricate genetic architecture of Pantoea agglomerans, revealing its adaptability to inhabit diverse niches and thrive in varied environments.

One-step cloning and targeted duplication of Pantoea ananatis chromosomal fragments.

In this study, we describe a novel method for one-step cloning and targeted duplication of P. ananatis chromosomal fragments. According to this method, the chromosomal region of interest is subcloned in vivo via λ Red recombination into the short synthetic non-replicable DNA fragment containing the excisable antibiotic-resistance marker gene and φ80 att-P site. The resulting circular non-replicating DNA molecule was immediately inserted into an alternative chromosomal locus due to φ80-integrase activity. To this end, the specially designed helper plasmid pONI, which can provide both the λ Red recombineering and φ80-integrase-mediated insertion, was constructed. In the described method, PCR amplification of the cloning fragment is unnecessary, making it convenient for manipulation of long-length DNA. Additionally, the possibility of spontaneous mutations occurring is completely precluded. This method was effectively used for the targeted chromosomal integration of additional copies of individual genes and operons up to 16 kb in size.

Interference of AHL signal production in the phytophatogen Pantoea agglomerans as a sustainable biological strategy to reduce its virulence.

Pantoea agglomerans is considered one of the most ubiquitous and versatile organisms that include strains that induce diseases in various crops and occasionally cause opportunistic infections in humans. To develop effective strategies to mitigate its impact on plant health and agricultural productivity, a comprehensive investigation is crucial for better understanding its pathogenicity. One proposed eco-friendly approach involves the enzymatic degradation of quorum sensing (QS) signal molecules like N-acylhomoserine lactones (AHLs), known as quorum quenching (QQ), offering potential treatment for such bacterial diseases. In this study the production of C4 and 3-oxo-C6HSL was identified in the plant pathogenic P. agglomerans CFBP 11141 and correlated to enzymatic activities such as amylase and acid phosphatase. Moreover, the heterologous expression of a QQ enzyme in the pathogen resulted in lack of AHLs production and the attenuation of the virulence by mean of drastically reduction of soft rot disease in carrots and cherry tomatoes. Additionally, the interference with the QS systems of P. agglomerans CFBP 11141 by two the plant growth-promoting and AHL-degrading bacteria (PGP-QQ) Pseudomonas segetis P6 and Bacillus toyonensis AA1EC1 was evaluated as a potential biocontrol approach for the first time. P. segetis P6 and B. toyonensis AA1EC1 demonstrated effectiveness in diminishing soft rot symptoms induced by P. agglomerans CFBP 11141 in both carrots and cherry tomatoes. Furthermore, the virulence of pathogen notably decreased when co-cultured with strain AA1EC1 on tomato plants.

Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

Hitching a Ride in the Phyllosphere: Surfactant Production of Pseudomonas spp. Causes Co-swarming of Pantoea eucalypti 299R.

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non-yeast signal peptide-mediated cell surface display.

BACKGROUND: Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. RESULTS: In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi-site integrating genes into chromosome. The final strain gained 451.7 g L-1 isomaltulose with a conversion rate of 90.3% and a space-time yield of 50.2 g L-1 h-1. CONCLUSION: The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole-cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry.

Simultaneous removal of aliphatic and aromatic crude oil hydrocarbons by Pantoea agglomerans isolated from petroleum-contaminated soil in the west of Iran.

Hydrocarbons are considered as one of the most common and harmful environmental pollutants affecting human health and the environment. Bioremediation as an environmentally friendly, highly efficient, and cost-effective method in remediating oil-contaminated environments has been interesting in recent decades. In this study, hydrocarbon degrader bacterial strains were isolated from the highly petroleum-contaminated soils in the Dehloran oil field in the west of Iran. Out of 37 isolates, 15 can grow on M9 agar medium that contains 1.5 g L-1 of crude oil as the sole carbon source. The morphological, biochemical, and 16SrRNA sequencing analyses were performed for the isolates. The choosing of the isolates as the hydrocarbon degrader was examined by evaluating the efficacy of their crude oil removal at a concentration of 10 g L-1 in an aqueous medium. The results showed that five isolates belonging to Pseudomonas sp., Pseudomonas oryzihabitans, Roseomonas aestuarii, Pantoea agglomerans, and Arthrobacter sp. had a hyper hydrocarbon-degrading activity and they could remove more than 85% of the total petroleum hydrocarbon (TPH) after 96 h. The highest TPH removal of about 95.75% and biodegradation rate of 0.0997 g L-1 h-1 was observed for P. agglomerans. The gas chromatography-mass spectroscopy (GC-MS) analysis was performed during the biodegradation process by P. agglomerans to detect the degradation intermediates and final products. The results confirmed the presence of intermediates such as alcohols and fatty acids in the terminal oxidation pathway of alkanes in this biodegradation process. A promising P. agglomerans NB391 strain can remove aliphatic and aromatic hydrocarbons simultaneously.

Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).

Plant growth promoting capability and genetic diversity of bacteria isolated from mud volcano and lime cave of Andaman and Nicobar Islands

Twenty four bacterial strains from four different regions of mud volcano and lime cave were isolated to estimate their diversity, plant growth promoting and biocontrol activities to use them as inoculant strains in the fields. An excellent antagonistic effect against four plant pathogens and plant growth promoting properties such as IAA production, HCN production, phosphate solubilization, siderophore production, starch hydrolysis and hydrolytic enzymes syntheses were identified in OM5 (Pantoea agglomerans) and EM9 (Exiguobacterium sp.) of 24 studied isolates. Seeds (Chili and tomato) inoculation with plant growth promoting strains resulted in increased percentage of seedling emergence, root length and plant weight. Results indicated that co-inoculation gave a more pronounced effects on seedling emergence, secondary root numbers, primary root length and stem length, while inoculation by alone isolate showed a lower effect. Our results suggest that the mixed inocula of OM5 and EM9 strains as biofertilizers could significantly increase the production of food crops in Andaman archipelago by means of sustainable and organic agricultural system.

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51 percent and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.