Complex third-party effects in the Dictyostelium-Paraburkholderia symbiosis: prey bacteria that are eaten, carried or left behind.

Symbiotic interactions may change depending on third parties like predators or prey. Third-party interactions with prey bacteria are central to the symbiosis between Dictyostelium discoideum social amoeba hosts and Paraburkholderia bacterial symbionts. Symbiosis with inedible Paraburkholderia allows host D. discoideum to carry prey bacteria through the dispersal stage where hosts aggregate and develop into fruiting bodies that disperse spores. Carrying prey bacteria benefits hosts when prey are scarce but harms hosts when prey bacteria are plentiful, possibly because hosts leave some prey bacteria behind while carrying. Thus, understanding benefits and costs in this symbiosis requires measuring how many prey bacteria are eaten, carried and left behind by infected hosts. We found that Paraburkholderia infection makes hosts leave behind both symbionts and prey bacteria. However, the number of prey bacteria left uneaten was too small to explain why infected hosts produced fewer spores than uninfected hosts. Turning to carried bacteria, we found that hosts carry prey bacteria more often after developing in prey-poor environments than in prey-rich ones. This suggests that carriage is actively modified to ensure hosts have prey in the harshest conditions. Our results show that multi-faceted interactions with third parties shape the evolution of symbioses in complex ways.

The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

Paraburkholderia translucens sp. nov. and Paraburkholderia sejongensis sp. nov., isolated from grassland soil and showing plant growth promoting potential.

Two Gram-negative bacterial strains designated MMS20-SJTN17T and MMS20-SJTR3T were isolated from a grassland soil sample, and taxonomically characterized using a polyphasic approach. The 16S rRNA gene sequence analysis indicates that both strains belong to the genus Paraburkholderia of the class Betaproteobacteria, with strain MMS20-SJTN17T being mostly related to Paraburkholderia sprentiae WSM5005T (96.45 % sequence similarity) and strain MMS20-SJTR3T to Paraburkholderia tuberum STM678T (98.59 % sequence similarity). MMS20-SJTN17T could grow at 15-40 °C (optimum, 25-30 °C) and at pH 6.0-8.0 (optimum, pH 6.0-7.0), whereas MMS20-SJTR3T could grow at 10-40 °C (optimum, 30-37 °C) and at pH 6.0-8.0 (optimum, pH 6.0). Both strains tolerated up to 1 % (w/v) NaCl (optimum, 0 %). The major fatty acids of MMS20-SJTN17T were C16 : 0 and C19 : 0 cyclo ω8c, and those of MMS20-SJTR3T were C17 : 0 cyclo and a summed feature comprising C18 : 1 ω7c and/or C18 : 1 ω6c. The major isoprenoid quinone of both strains was ubiquinone-8 and the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Regarding plant growth promoting potential, both strains were capable of producing indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase, and also showed phosphate-solubilizing activity. A genome-based comparison using orthologous average nucleotide identity and digital DNA-DNA hybridization values indicates that strain MMS20-SJTN17T shares highest relatedness with Paraburkholderia monticola JC2948T and MMS20-SJTR3T with Paraburkholderia antibiotica G-4-1-8T, with values clearly below the cutoffs for species distinction. Examination of biosynthetic gene clusters responsible for secondary metabolite production reveals unique characteristics distinguishing each strain from closely related Paraburkholderia species. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenomic data, each strain should be classified as a novel species of the genus Paraburkholderia, for which the names Paraburkholderia translucens sp. nov. (=MMS20-SJTN17T=LMG 32366T=KCTC 82783T) and Paraburkholderia sejongensis sp. nov. (=MMS20-SJTR3T=LMG 32367T=KCTC 82784T) are proposed.

Paraburkholderia largidicola sp. nov., a gut symbiont of the bordered plant bug Physopelta gutta.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated strains F2T and PGU16, were isolated from the midgut crypts of the bordered plant bug Physopelta gutta, collected in Okinawa prefecture, Japan. Although these strains were derived from different host individuals collected at different times, their 16S rRNA gene sequences were identical and showed the highest similarity to Paraburkholderia caribensis MWAP64T (99.3 %). The genome of strain F2T consisted of two chromosomes and two plasmids, and its size and G+C content were 9.28 Mb and 62.4 mol% respectively; on the other hand, that of strain PGU16 consisted of two chromosomes and three plasmids, and its size and G+C content were 9.47 Mb and 62.4 mol%, respectively. Phylogenetic analyses revealed that these two strains are members of the genus Paraburkholderia. The digital DNA-DNA hybridization value between these two strains was 92.4 %; on the other hand, the values between strain F2T and P. caribensis MWAP64T or phylogenetically closely related Paraburkholderia species were 44.3 % or below 49.1 %. The predominant fatty acids of both strains were C16 : 0, C17 : 0 cyclo, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), and C19 : 0 cyclo ω8c, and their respiratory quinone was ubiquinone 8. Based on the above genotypic and phenotypic characteristics, strains F2T and PGU16 represent a novel species of the genus Paraburkholderia for which the name Paraburkholderia largidicola sp. nov. is proposed. The type strain is F2T (=NBRC 115765T=LMG 32765T).

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.

Exploring the biocontrol potential of rocket (Eruca sativa) extracts and associated microorganisms against Verticillium wilt.

AIMS: This study aimed to assess the impact of rocket (Eruca sativa) extract on Verticillium wilt in eggplants, explore rhizospheric microorganisms for disease biocontrol, and evaluate selected strains' induced systemic resistance (ISR) potential while characterizing their genomic and biosynthetic profiles. METHODS AND RESULTS: Rocket extract application led to a significant reduction in Verticillium wilt symptoms in eggplants compared to controls. Isolated microorganisms from treated soil, including Paraburkholderia oxyphila EP1, Pseudomonas citronellolis EP2, Paraburkholderia eburnea EP3, and P. oxyphila EP4 and EP5, displayed efficacy against Verticillium dahliae, decreasing disease severity and incidence in planta. Notably, strains EP3 and EP4 triggered ISR in eggplants against V. dahliae. Genomic analysis unveiled shared biosynthetic gene clusters, such as ranthipeptide and non-ribosomal peptide synthetase-metallophore types, among the isolated strains. Additionally, metabolomic profiling of EP2 revealed the production of metabolites associated with amino acid metabolism, putative antibiotics, and phytohormones. CONCLUSIONS: The application of rocket extract resulted in a significant reduction in Verticillium wilt symptoms in eggplants, while the isolated microorganisms displayed efficacy against V. dahliae, inducing systemic resistance and revealing shared biosynthetic gene clusters, with metabolomic profiling highlighting potential disease-suppressing metabolites.

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.

Visualization of root extracellular traps in an ectomycorrhizal woody plant (Pinus densiflora) and their interactions with root-associated bacteria.

MAIN CONCLUSION: Extracellular traps in the primary root of Pinus densiflora contribute to root-associated bacterial colonization. Trapped rhizobacteria induce the production of reactive oxygen species in root-associated, cap-derived cells. Ectomycorrhizal (ECM) woody plants, such as members of Pinaceae and Fagaceae, can acquire resistance to biotic and abiotic stresses through the formation of mycorrhiza with ECM fungi. However, germinated tree seedlings do not have mycorrhizae and it takes several weeks for ectomycorrhizae to form on their root tips. Therefore, to confer protection during the early growth stage, bare primary roots require defense mechanisms other than mycorrhization. Here, we attempted to visualize root extracellular traps (RETs), an innate root defense mechanism, in the primary root of Pinus densiflora and investigate the interactions with root-associated bacteria isolated from ECM and fine non-mycorrhizal roots. Histological and histochemical imaging and colony-forming unit assays demonstrated that RETs in P. densiflora, mainly consisting of root-associated, cap-derived cells (AC-DCs) and large amounts of root mucilage, promote bacterial colonization in the rhizosphere, despite also having bactericidal activity via extracellular DNA. Four rhizobacterial strains retarded the mycelial growth of a pathogenic strain belonging to the Fusarium oxysporum species complex in dual culture assay. They also induced the production of reactive oxygen species (ROS) from host tree AC-DCs without being excluded from the rhizosphere of P. densiflora. Applying three Paraburkholderia strains, especially PM O-EM8 and PF T-NM22, showed significant differences in the ROS levels from the control group. These results reveal the indirect contributions of rhizobacteria to host root defense and suggest that root-associated bacteria could be a component of RETs as a first line of defense against root pathogens in the early growth stage of ECM woody plants.

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.

Isolation of new Paraburkholderia strains for polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are bioplastics that can serve as substitutes for petroleum-based plastics with the advantages of being biodegradable, biocompatible, and biobased. The microbial production of polyhydroxyalkanoates is generally conducted in the presence of sugar mixes rich in monosaccharides. In this study, molecular and cultural approaches based on forest soils enriched with hydrocarbon complexes led to the identification and isolation of microbial strains affiliated with Paraburkholderia sp. that dominated the microbial communities that are recognized among the top polyhydroxyalkanoates producers. The genome sequencing of those isolated affiliated strains showed that compared to the reference type strain of their species, they harbored more gene copies of the enzymes involved in PHB synthesis. The microbial conversion of sugar mixes for the newly isolated strains showed a higher PHB production (g/L) and content (%) than was exhibited by the reference strain type of that genus Paraburkholderia for PHB production (P. sacchari LMG 19450T).

Complexities of Inferring Symbiont Function: Paraburkholderia Symbiont Dynamics in Social Amoeba Populations and Their Impacts on the Amoeba Microbiota.

The relationship between the social amoeba Dictyostelium discoideum and its endosymbiotic bacteria Paraburkholderia provides a model system for studying the development of symbiotic relationships. Laboratory experiments have shown that any of three species of the Paraburkholderia symbiont allow D. discoideum food bacteria to persist through the amoeba life cycle and survive in amoeba spores rather than being fully digested. This phenomenon is termed "farming," as it potentially allows spores dispersed to food-poor locations to grow their own. The occurrence and impact of farming in natural populations, however, have been a challenge to measure. Here, we surveyed natural D. discoideum populations and found that only one of the three symbiont species, Paraburkholderia agricolaris, remained prevalent. We then explored the effect of Paraburkholderia on the amoeba microbiota, expecting that by facilitating bacterial food carriage, it would diversify the microbiota. Contrary to our expectations, Paraburkholderia tended to infectiously dominate the D. discoideum microbiota, in some cases decreasing diversity. Similarly, we found little evidence for Paraburkholderia facilitating the carriage of particular food bacteria. These findings highlight the complexities of inferring symbiont function in nature and suggest the possibility that Paraburkholderia could be playing multiple roles for its host. IMPORTANCE The functions of symbionts in natural populations can be difficult to completely discern. The three Paraburkholderia bacterial farming symbionts of the social amoeba Dictyostelium discoideum have been shown in the laboratory environment to allow the amoebas to carry, rather than fully digest, food bacteria. This potentially provides a fitness benefit to the amoebas upon dispersal to food-poor environments, as they could grow their food. We expected that meaningful food carriage would manifest as a more diverse microbiota. Surprisingly, we found that Paraburkholderia tended to infectiously dominate the D. discoideum microbiota rather than diversifying it. We determined that only one of the three Paraburkholderia symbionts has increased in prevalence in natural populations in the past 20 years, suggesting that this symbiont may be beneficial, however. These findings suggest that Paraburkholderia may have an alternative function for its host, which drives its prevalence in natural populations.

Differential Genetic Strategies of Burkholderia vietnamiensis and Paraburkholderia kururiensis for Root Colonization of Oryza sativa subsp. japonica and O. sativa subsp. indica, as Revealed by Transposon Mutagenesis Sequencing.

Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCEBurkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.

Paraburkholderia bengalensis sp. nov. isolated from roots of Oryza sativa, IR64.

Paraburkholderia bengalensis sp. nov. strain IR64_4_BI was isolated from rice roots cultivated in Madhyamgram field station of Bose Institute, West Bengal, India. IR64_4_BI is a Gram-negative, motile, nitrate-reducing, nitrogen-fixing bacterium. Whole-cell fatty acid analyses of IR64_4_BI show C16:0, summed feature 8 (comprising C18:1ω7c and/or C18:1 ω 6c) and summed feature 3(C16:1 w7c/C16:1 w6c or C16:1 ω 7c/C16:1 ω 6c) were the predominant fatty acids. 16S rRNA phylogeny showed that it was most similar to P. phymatum STM815T (98.5% identity), P. terrae KMY02T (98.44% identity) and P. hospita LMG 20598T (98.32% identity). The Average Nucleotide Identity-BLAST (ANIb) of P. bengalensis IR64_4_BI with P. hospita DSM 17164T, P. terrae DSM 17804T, P. phymatum STM815T and P. hospita LMG 20598T was 83.11, 83.52, 84.5 and 83.12% respectively. Comparison of genome sequence of IR64_4_BI with other species of Paraburkholderia using the Multi-locus species tree software show that P. bengalensis IR64_4_BI is a novel species. The ability of P. bengalensis IR64_4_BI to survive on nitrogen-free medium under microaerophilic conditions and the abundance of nitrogen metabolism-related genes makes this strain a potential candidate for developing a nitrogen-fixing system in rice. Based on genotypic, phenotypic and chemotaxonomic studies, we propose that IR64_4_BI (= MTCC 13051 = JCM 34777) is a new species of Paraburkholderia which has been assigned as Paraburkholderia bengalensis sp.nov.

Aerobic Bioaugmentation to Decrease Polychlorinated Biphenyl (PCB) Emissions from Contaminated Sediments to Air.

We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2'-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.

Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64.

Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription-polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400.

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic-degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H2O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZoxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H2O2. The effects of these oxidants on gene expression (qRT-PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR, ahpC, ahpF, kat, trxB, dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli. The LB400 genome also harbors the soxR, fumC, acnA, sodB, fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli. The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa ΔsoxR. Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR, ahpC1, katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR, fumC, ahpC1, sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR-regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

Tracking and plant growth-promoting effect of Paraburkholderia tropica MTo-293 applied to Solanum lycopersicum.

Paraburkholderia tropica MTo-293 was applied as an experimental bio-input to Solanum lycopersicum (tomato) cv. Platense. Different plant growth systems and inoculation strategies were tested to evaluate P. tropica plant colonization at the seedling stage (growth chamber) using culture-dependent and -independent techniques. The effect of P. tropica on plant growth was evaluated in the growth chamber and greenhouse (productive stage) by biomass accumulation and fruit production, respectively. P. tropica was able to colonize the surface and inner root and stem of tomato seedlings regardless of the inoculation strategy-at sowing and/or before transplanting-showing the competitive nature of P. tropica in nonsterile substrate systems. A nested polymerase chain reaction was validated to track P. tropica in tomato plants even in the inner stem with endophytic P. tropica populations of less than 102 CFU g-1 of fresh weight. Efficient colonization of P. tropica correlated with a positive effect on tomato growth when applied at sowing and/or before transplanting: plant growth promotion was observed not only at the seedling stage but also at productive stages improving crop yield in two different seasons. To our knowledge, this report is the first to track and evaluate the plant growth-promoting effect of P. tropica MTo-293 in tomato plants grown in nonsterile substrate systems.

Multiple Copies of flhDC in Paraburkholderia unamae Regulate Flagellar Gene Expression, Motility, and Biofilm Formation.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia, and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1, plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter-lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia. In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2. The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae, and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC. In P. unamae, the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

Proteomic analysis reveals how pairing of a Mycorrhizal fungus with plant growth-promoting bacteria modulates growth and defense in wheat.

Plants rely on their microbiota for improving the nutritional status and environmental stress tolerance. Previous studies mainly focused on bipartite interactions (a plant challenged by a single microbe), while plant responses to multiple microbes have received limited attention. Here, we investigated local and systemic changes induced in wheat by two plant growth-promoting bacteria (PGPB), Azospirillum brasilense and Paraburkholderia graminis, either alone or together with an arbuscular mycorrhizal fungus (AMF). We conducted phenotypic, proteomic, and biochemical analyses to investigate bipartite (wheat-PGPB) and tripartite (wheat-PGPB-AMF) interactions, also upon a leaf pathogen infection. Results revealed that only AMF and A. brasilense promoted plant growth by activating photosynthesis and N assimilation which led to increased glucose and amino acid content. The bioprotective effect of the PGPB-AMF interactions on infected wheat plants depended on the PGPB-AMF combinations, which caused specific phenotypic and proteomic responses (elicitation of defense related proteins, immune response and jasmonic acid biosynthesis). In the whole, wheat responses strongly depended on the inoculum composition (single vs. multiple microbes) and the investigated organs (roots vs. leaf). Our findings showed that AMF is the best-performing microbe, suggesting its presence as the crucial one for synthetic microbial community development.

Paraburkholderia dioscoreae sp. nov., a novel plant associated growth promotor.

A novel bacterium, designated strain Msb3T, was recently isolated from leaves of the yam family plant Dioscorea bulbifera (Dioscoreaceae). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Paraburkholderia with Paraburkholderia xenovorans as nearest validly named neighbour taxon (99.3 % sequence similarity towards the P. xenovorans type strain). Earlier genome sequence analysis revealed a genome of 8.35 Mb in size with a G+C content of 62.5 mol%, which was distributed over two chromosomes and three plasmids. Here, we confirm that strain Msb3T represents a novel Paraburkholderia species. In silico DNA-DNA hybridization and average nucleotide identity (OrthoANIu) analyses towards P. xenovorans LB400T yielded 58.4 % dDDH and 94.5 % orthoANIu. Phenotypic and metabolic characterization revealed growth at 15 °C on tryptic soy agar, growth in the presence of 1 % NaCl and the lack of assimilation of phenylacetic acid as distinctive features. Together, these data demonstrate that strain Msb3T represents a novel species of the genus Paraburkholderia, for which we propose the name Paraburkholderia dioscoreae sp. nov. The type strain is Msb3T (=LMG 31881T, DSM 111632T, CECT 30342T).