The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Keguizhuia sedimenti gen. nov., sp. nov., isolated from river sediment represents a novel genus of the family Oxalobacteraceae.

A Gram-stain-negative bacterium, designated as R-40T, was isolated from sediment of the Mulong river in Mianyang city, Sichuan province, PR China. The cells of strain R-40T were aerobic non-motile and formed translucent white colonies on R2A agar. Growth occurred at 15-37 °C (optimum 30 °C), pH 5.0-9.0 (optimum 7.0) and salinities of 0-3.0 % (w/v, optimum 0 %). R-40T showed 95.2-96.6 % 16S rRNA gene sequence similarities with the type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum in the family Oxalobacteraceae. The results of phylogenetic analysis based on genome sequences indicated that the strain was clustered with type strains of species of the genera Oxalicibacterium and Herminiimonas in the family Oxalobacteraceae but formed a distinct lineage. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and average amino acid identity (AAI) values between R-40T and type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum ranged from 69.3 to 74.1 %, from 18.2 to 21.4 % and from 60.1 to 67.4 %, respectively. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major quinone was ubiquinone-8 (Q-8). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and small amounts of glycophospholipids. The genome size of R-40T was 5.1 Mbp with 54.0 % DNA G+C content. On the basis of the evidence presented in this study, strain R-40T represents a novel species of a novel genus in the family Oxalobacteraceae, for which the name Keguizhuia sedimenti gen. nov., sp. nov. (type strain R-40T=MCCC 1K08818T=KCTC 8137T) is proposed.

Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect.

Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.

Black Carbon Impacts on Paraburkholderia xenovorans Strain LB400 Cell Enrichment and Activity: Implications toward Lower-Chlorinated Polychlorinated Biphenyls Biodegradation Potential.

Volatilization of lower-chlorinated polychlorinated biphenyls (LC-PCBs) from sediment poses health threats to nearby communities and ecosystems. Biodegradation combined with black carbon (BC) materials is an emerging bioaugmentation approach to remove PCBs from sediment, but development of aerobic biofilms on BC for long-term, sustained LC-PCBs remediation is poorly understood. This work aimed to characterize the cell enrichment and activity of biphenyl- and benzoate-grown Paraburkholderia xenovorans strain LB400 on various BCs. Biphenyl dioxygenase gene (bphA) abundance on four BC types demonstrated corn kernel biochar hosted at least 4 orders of magnitude more attached cells per gram than other feedstocks, and microscopic imaging revealed the attached live cell fraction was >1.5× more on corn kernel biochar than GAC. BC characteristics (i.e., sorption potential, pore size, pH) appear to contribute to cell attachment differences. Reverse transcription qPCR indicated that BC feedstocks significantly influenced bphA expression in attached cells. The bphA transcript-per-gene ratio of attached cells was >10-fold more than suspended cells, confirmed by transcriptomics. RNA-seq also demonstrated significant upregulation of biphenyl and benzoate degradation pathways on attached cells, as well as revealing biofilm formation potential/cell-cell communication pathways. These novel findings demonstrate aerobic PCB-degrading cell abundance and activity could be tuned by adjusting BC feedstocks/attributes to improve LC-PCBs biodegradation potential.

Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis.

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC-MS and NMR. KEY POINTS: • A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified. • The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum. • Synthesis of acyl amino acids was achieved in good yields.

Parachitinimonas caeni gen. nov., sp. nov., a novel member of family Burkholderiaceae isolated from activated sludge collected in Shenzhen, PR China.

A Gram-stain-negative, strictly aerobic and filamentous bacterial strain, designated as DQS-5T, was isolated from the activated sludge of a municipal sewage treatment plant in Shenzhen, PR China. Optimal growth was observed at 28 °C and pH 7.5. Catalase and oxidase activities were detected. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DQS-5T was most closely related to the genera Chitinimonas and Chitinivorax (91.0-93.4 % and 92.5 % 16S rRNA gene sequence similarity, respectively) and was close to the member of the family Burkholderiaceae. The complete genome sequence of strain DQS-5T contains 5 653 844 bp and 57.3 mol% G+C. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between the genome of strain DQS-5T and those of its close relatives were 75.9-77.2, 19.0-20.3 and 57.2-61.8 %, respectively. Chemotaxonomic analysis of strain DQS-5T indicated that the sole respiratory quinone was ubiquinone-8, the predominant cellular fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and the major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid and aminolipid. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrate that strain DQS-5T represents a novel species in a novel genus within the family Burkholderiaceae, for which the name Parachitinimonas caeni gen. nov., sp. nov., is proposed. Strain DQS-5T (=KCTC 92788T=CCTCC AB 2022320T) is the type and only strain of P. caeni.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Responses of a polycyclic aromatic hydrocarbon-degrading bacterium, Paraburkholderia fungorum JT-M8, to Cd (II) under P-limited oligotrophic conditions.

For the bioremediation of mixed-contamination sites, studies on polycyclic aromatic hydrocarbon (PAH) degradation or Cd (II) tolerance in bacteria are commonly implemented in nutrient-rich media. In contrast, in the field, inocula usually encounter harsh oligotrophic habitats. In this study, the environmental strain Paraburkholderia fungorum JT-M8 was used to explore the overlooked Cd (II) defense mechanism during PAH dissipation under P-limited oligotrophic condition. The results showed that the growth and PAH degradation ability of JT-M8 under Cd (II) stress were correlated with phosphate contents and exhibited self-regulating properties. Phosphates mainly affected the Cd (II) content in solution, while the cellular distribution of Cd (II) depended on Cd (II) levels; Cd (II) was mainly located in the cytoplasm when exposed to less Cd (II), and vice versa. The unique Cd (II) detoxification pathways could be classified into three aspects: (i) Cd (II) ionic equilibrium and dose-response effects regulated by environmental matrices (phosphate contents); (ii) bacterial physiological self-regulation, e.g., cell surface-binding, protein secretion and active transport systems; and (iii) specific adaptive responses (flagellum aggregation). This study emphasizes the importance of considering culture conditions when assessing the metal tolerance and provides new insight into the bacterial detoxification process of complex PAH-Cd (II) pollutants.

Effects of biochar layer position on treatment performance and microbial community in subsurface flow constructed wetlands for removal of cadmium and lead.

Levels of cadmium (Cd) and lead (Pb) correspond to common composition in acid mine wastewater of Hunan Province of China. The removal path of Cd and Pb and the structure of microbial community were investigated by developing constructed wetlands (CWs) with different layer positions of biochar. The biochar as a layer at the bottom of CW (BCW) system exhibited maximum Cd and Pb removal efficiencies of 96.6-98.6% and 97.2-98.9%, respectively. Compared with original soil, BCW increased the relative proportions of Proteobacteria, Firmicutes, Acidobacteriota, Verrucomicrobiota, Desulfobacterota, Armatimonadota, Bacteroidota, Patescibacteria, Basidiomycota (phylum level) and Burkholderia-Caballeronia-Paraburkholderia, Citrifermentans, Chthonomonadales, Cellulomonas, Geothrix, Terracidiphilus, Gallionellaceae, Microbacterium, Vanrija, Apiotrichum, Saitozyma, Fusarium (genus level). The concentrations of Cd and Pb were positively correlated with the abundance of Verrucomicrobiota, Basidiomycota (phylum level), and Methylacidiphilaceae, Meyerozyma, Vanrija (genus level). This study demonstrates that BCW system can improve removal performance toward Cd and Pb, as well as alter microbial community.

Trinickia violacea sp. nov. and Trinickia terrae sp. nov., isolated from forest soil.

Two Gram-stain negative, aerobic and rod-shaped bacterial strains, DHOD12T and 7GSK02T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain DHOD12T grew at 4-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.5-6.5) and in the presence of 0-1.5 % (w/v; optimum, 0-0.5 %)NaCl; while strain 7GSK02T grew at 12-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.0-6.0) and in the presence of 0-0.5 % (w/v; optimum, 0 %) NaCl. Strains DHOD12T and 7GSK02T had the highest 16S rRNA sequence similarities of 98.0 and 98.3 % with the same species Trinickia mobilis DHG64T, respectively, and 98.4 % between themselves. In the 16S rRNA phylogeny, they formed a clade that was sister to a major cluster consisting of all described Trinickia species. Phylogenomic analyses with the UBCG and PhyloPhlAn methods consistently showed that strains DHOD12T and 7GSK02T formed a clade with T. mobilis DHG64T that was a sister of a cluster containing the remainder of the Trinickia species. The DNA G+C contents of strains DHOD12T and 7GSK02T were 63.1 and 64.6 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity values of strains DHOD12T, 7GSK02T and their closely related strains were in the ranges of 21.6-31.4 % and 77.1-86.9 %, respectively. These two strains had the same major respiratory quinone, ubiquinone-8, and both had C16 : 0, C17 : 0 cyclo and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as their major fatty acids. Their major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Genomic analysis indicated that the two strains could have the potential to degrade aromatic compounds like other Trinickia species. On the basis of phenotypic and phylogenetic results, strains DHOD12T and 7GSK02T represent two novel species of the genus Trinickia, for which the names Trinickia violacea sp. nov. (type strain DHOD12T=LMG 30258T=CGMCC 1.15436T) and Trinickia terrae sp. nov. (type strain 7GSK02T=CGMCC 1.15432T=KCTC 62468T) are proposed.

Outbreak of Pandoraea commovens Infections among Non-Cystic Fibrosis Intensive Care Patients, Germany, 2019-2021.

Pandoraea spp. are gram-negative, nonfermenting rods mainly known to infect patients with cystic fibrosis (CF). Outbreaks have been reported from several CF centers. We report a Pandoraea spp. outbreak comprising 24 non-CF patients at a large university hospital and a neighboring heart center in Germany during July 2019-December 2021. Common features in the patients were critical illness, invasive ventilation, antimicrobial pretreatment, and preceding surgery. Complicated and relapsing clinical courses were observed in cases with intraabdominal infections but not those with lower respiratory tract infections. Genomic analysis of 15 isolates identified Pandoraea commovens as the genetically most similar species and confirmed the clonality of the outbreak strain, designated P. commovens strain LB-19-202-79. The strain exhibited resistance to most antimicrobial drugs except ampicillin/sulbactam, imipenem, and trimethoprim/sulfamethoxazole. Our findings suggest Pandoraea spp. can spread among non-CF patients and underscore that clinicians and microbiologists should be vigilant in detecting and assessing unusual pathogens.

Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi.

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

Pioneering Desmodium spp. are nodulated by natural populations of stress-tolerant alpha- and beta-rhizobia.

The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions.

Species diversity of environmentally-transmitted bacteria colonizing Riptortus pedestris (Hemiptera: Alydidae) and symbiotic effects of the most dominant bacteria.

Riptortus pedestris (Hemiptera: Alydidae) establish endosymbiosis with specific bacteria from extremely diverse microbiota in soil. To better understand ecology and evolution of the symbiosis, it is important to characterize bacterial species diversity colonizing R. pedestris and evaluate their symbiotic effects. Nonetheless, previous research was limited to a few bacteria strains such as Caballeronia insecticola. In this study, second-instar nymphs were provided with field soils and reared to adult. Then, bacteria colonizing the midgut M4 region of R. pedestris were analyzed for bacterial species identification based on the 16S rRNA gene. First, a total of 15 bacterial species were detected belonging to Burkholderiaceae. Most of R. pedestris were found to harbor single bacterial species, whereas several insects harbored at most two bacterial species simultaneously. Among the total insects harboring single bacterial species, 91.2% harbored genus Caballeronia. The most dominant species was C. jiangsuensis, not previously documented for symbiotic associations with R. pedestris. Second, in laboratory conditions, C. jiangsuensis significantly enhanced the development, body size, and reproductive potentials of R. pedestris, compared to individuals with no symbiotic bacteria. These results add novel information to better understand symbiotic bacteria community establishing in R. pedestris and symbiotic effects on the host insects.

Paraburkholderia flagellata sp. nov. and Paraburkholderia adhaesiva sp. nov., two novel species isolated from forest soil in Dinghushan Biosphere Reserve in Guangdong, China.

Two Gram-stain-negative, aerobic, motile and short rod strains, designated 4D117T and ZD32-2T, were isolated from the forest soils. Strains 4D117T and ZD32-2T grew optimally at pH 4.0-6.5, 20-33 °C and pH 4.5-7.0, 33 °C, respectively, and both at 0.5% (w/v) NaCl concentration. Strains 4D117T and ZD32-2T shared the highest 16S rRNA gene sequence similarity with P. acidiphila 7Q-K02T (99.1%) and P. ferrariae NBRC 106233T (98.7%), respectively. The genome size and G + C contents of strains 4D117T and ZD32-2T were 9,002,095 bp, 62.9% and 6,974,420 bp, 61.7%, respectively. The dDDH and ANI values between strains 4D117T, ZD32-2T and closely related Paraburkholderia species were in the ranges of 21.9-51.6% and 82.9-94.4%, and 81.7% and 25.4% between themself, respectively. Functional genomic analysis showed both strains were capable of degrading contaminants, such as benzoate, anthranilic acid and catechol for 4D117T, and benzene and catechol for ZD32-2T, indicating that they may have potentials for soil pollutant treatment. The main polar lipids of strains 4D117T and ZD32-2T were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain 4D117T contained C16:0, C19:0 cyclo ω8c and C18:1 ω7c and/or C18:1 ω6c, while strain ZD32-2T had C16:0 and C17:0 cyclo as their major cellular fatty acids (> 10%). Based on the phenotypic characters and genomic data, strains 4D117T and ZD32-2T represent two novel species of genus Paraburkholderia, for which the names Paraburkholderia flagellata sp. nov. (type strain 4D117T = GDMCC 1.2617T = NBRC 115278T) and Paraburkholderia adhaesiva sp. nov. (type strain ZD32-2T = GDMCC 1.2622T = NBRC 115282T) are proposed.

Metagenomics of African Empogona and Tricalysia (Rubiaceae) reveals the presence of leaf endophytes.

Background: Leaf symbiosis is a phenomenon in which host plants of Rubiaceae interact with bacterial endophytes within their leaves. To date, it has been found in around 650 species belonging to eight genera in four tribes; however, the true extent in Rubiaceae remains unknown. Our aim is to investigate the possible occurrence of leaf endophytes in the African plant genera Empogona and Tricalysia and, if present, to establish their identity. Methods: Total DNA was extracted from the leaves of four species of the Coffeeae tribe (Empogona congesta, Tricalysia hensii, T. lasiodelphys, and T. semidecidua) and sequenced. Bacterial reads were filtered out and assembled. Phylogenetic analysis of the endophytes was used to reveal their identity and their relationship with known symbionts. Results: All four species have non-nodulated leaf endophytes, which are identified as Caballeronia. The endophytes are distinct from each other but related to other nodulated and non-nodulated endophytes. An apparent phylogenetic or geographic pattern appears to be absent in endophytes or host plants. Caballeronia endophytes are present in the leaves of Empogona and Tricalysia, two genera not previously implicated in leaf symbiosis. This interaction is likely to be more widespread, and future discoveries are inevitable.

Paraburkholderia symbionts isolated from Dictyostelium discoideum induce bacterial carriage in other Dictyostelium species.

The social amoeba Dictyostelium discoideum engages in a complex relationship with bacterial endosymbionts in the genus Paraburkholderia, which can benefit their host by imbuing it with the ability to carry prey bacteria throughout its life cycle. The relationship between D. discoideum and Paraburkholderia has been shown to take place across many strains and a large geographical area, but little is known about Paraburkholderia's potential interaction with other dictyostelid species. We explore the ability of three Paraburkholderia species to stably infect and induce bacterial carriage in other dictyostelid hosts. We found that all three Paraburkholderia species successfully infected and induced carriage in seven species of Dictyostelium hosts. While the overall behaviour was qualitatively similar to that previously observed in infections of D. discoideum, differences in the outcomes of different host/symbiont combinations suggest a degree of specialization between partners. Paraburkholderia was unable to maintain a stable association with the more distantly related host Polysphondylium violaceum. Our results suggest that the mechanisms and evolutionary history of Paraburkholderia's symbiotic relationships may be general within Dictyostelium hosts, but not so general that it can associate with hosts of other genera. Our work further develops an emerging model system for the study of symbiosis in microbes.

Paraburkholderia sabiae Uses One Type VI Secretion System (T6SS-1) as a Powerful Weapon against Notorious Plant Pathogens.

Paraburkholderia sabiae LMG24235 is a nitrogen-fixing betaproteobacterium originally isolated from a root nodule of Mimosa caesalpiniifolia in Brazil. We show here that this strain effectively kills strains from several bacterial families (Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae) which include important plant pathogens in a contact-dependent manner. De novo assembly of the first complete genome of P. sabiae using long sequencing reads and subsequent annotation revealed two gene clusters predicted to encode type VI secretion systems (T6SS), which we named T6SS-1 and T6SS-3 according to previous classification methods (G. Shalom, J. G. Shaw, and M. S. Thomas, Microbiology, 153:2689-2699, 2007, https://doi.org/10.1099/mic.0.2007/006585-0). We created P. sabiae with mutations in each of the two T6SS gene clusters that abrogated their function, and the T6SS-1 mutant was no longer able to outcompete other strains in a contact-dependent manner. Notably, our analysis revealed that T6SS-1 is essential for competition against several important plant pathogens in vitro, including Burkholderia plantarii, Ralstonia solanacearum, Pseudomonas syringae, and Pectobacterium carotovorum. The 9-log reduction in P. syringae cells in the presence of P. sabiae was particularly remarkable. Importantly, in an in vivo assay, P. sabiae was able to protect potato tubers from bacterial soft rot disease caused by P. carotovorum, and this protection was partly dependent on T6SS-1. IMPORTANCE Rhizobia often display additional beneficial traits such as the production of plant hormones and the acquisition of limited essential nutrients that improve plant growth and enhance plant yields. Here, we show that the rhizobial strain P. sabiae antagonizes important phytopathogens such as P. carotovorum, P. syringae, and R. solanacearum and that this effect is due to contact-dependent killing mediated by one of two T6SS systems identified in the complete, de novo assembled genome sequence of P. sabiae. Importantly, co-inoculation of Solanum tuberosum tubers with P. sabiae also resulted in a drastic reduction of soft rot caused by P. carotovorum in an in vivo model system. This result highlights the protective potential of P. sabiae against important bacterial plant diseases, which makes it a valuable candidate for application as a biocontrol agent. It also emphasizes the particular potential of rhizobial inoculants that combine several beneficial effects such as plant growth promotion and biocontrol for sustainable agriculture.

Trinickia mobilis sp. nov. and Trinickia acidisoli sp. nov., isolated from soil.

Two novel Gram-stain-negative, aerobic and rod-shaped bacterial strains, designated DHG64T and 4D114T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. DHG64T grew at 12-37 °C (optimum 33 °C), pH 4.5-10.0 (optimum 6.5-7.5) and in the presence of 0-2.0 % NaCl (w/v); while 4D114T grew at 12-37 °C (optimum 20-33 °C), pH 4.0-7.0 (optimum 4.5-6.0) and in the presence of 0-1.0 % NaCl (w/v). DHG64T and 4D114T showed 97.1-98.0 and 97.5-98.4 % 16S rRNA gene sequence similarities with seven species of the genus Trinickia with validly published names, respectively. In the phylogenetic trees based on 16S rRNA gene and genome sequences, both strains formed a clade with the members of genus Trinickia but well separated from each other. The average nucleotide identity and digital DNA-DNA hybridisation values for the novel strains to all species of the genus Trinickia with validly published names were in the ranges of 80.6-85.0 and 22.4-28.0 %, respectively. DHG64T contained C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c, while 4D114T had C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) as the major cellular fatty acids. The major polar lipids for strains DHG64T and 4D114T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C contents of DHG64T and 4D114T were 63.0 and 62.8 mol%, respectively. Genomic analyses indicated that DHG64T and 4D114T may have potential for various applications, such as developing drugs against certain health problems and restoring environments polluted with metal ions and/or benzoate. On the basis of the results of morphological, physiological, biochemical and phylogenetic analyses, strains DHG64T and 4D114T were classified as representing two novel species of the genus Trinickia, for which the names Trinickia mobilis sp. nov. (type strain DHG64T = KACC 21223T = GDMCC 1.1282T) and Trinickia acidisoli sp. nov. (type strain 4D114T = KCTC 82876T = GDMCC 1.2131T) are proposed.

The social amoeba Dictyostelium discoideum rescues Paraburkholderia hayleyella, but not P. agricolaris, from interspecific competition.

Bacterial endosymbionts can provide benefits for their eukaryotic hosts, but it is often unclear if endosymbionts benefit from these relationships. The social amoeba Dictyostelium discoideum associates with three species of Paraburkholderia endosymbionts, including P. agricolaris and P. hayleyella. These endosymbionts can be costly to the host but are beneficial in certain contexts because they allow D. discoideum to carry prey bacteria through the dispersal stage. In experiments where no other species are present, P. hayleyella benefits from D. discoideum while P. agricolaris does not. However, the presence of other species may influence this symbiosis. We tested if P. agricolaris and P. hayleyella benefit from D. discoideum in the context of resource competition with Klebsiella pneumoniae, the typical laboratory prey of D. discoideum. Without D. discoideum, K. pneumoniae depressed the growth of both Paraburkholderia symbionts, consistent with competition. P. hayleyella was more harmed by interspecific competition than P. agricolaris. We found that P. hayleyella was rescued from competition by D. discoideum, while P. agricolaris was not. This may be because P. hayleyella is more specialized as an endosymbiont; it has a highly reduced genome compared to P. agricolaris and may have lost genes relevant for resource competition outside of its host.