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.

Inhibitory activity of bacterial lipopeptides against Fusarium oxysporum f.sp. Strigae.

This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate whether the presence of bacteria inhibits fungal growth and whether this inhibition is due to the release of bacterial metabolites, particularly LP. Selected endophytic bacterial strains with known plant-growth promoting potential were cultured in the presence of Fusarium oxysporum f.sp. strigae (Fos), which was applied as model fungal organism. The extracellular metabolome of tested bacteria, with a focus on LP, was characterized, and the inhibitory effect of bacterial LP on fungal growth was investigated. The results showed that Bacillus velezensis GB03 and FZB42, as well as B. subtilis BSn5 exhibited the strongest antagonism against Fos. Paraburkholderia phytofirmans PsJN, on the other hand, tended to have a slight, though non-significant growth promotion effect. Crude LP from strains GB03 and FZB42 had the strongest inhibitory effect on Fos, with a significant inhibition of spore germination and damage of the hyphal structure. Liquid chromatography tandem mass spectrometry revealed the production of several variants of iturin, fengycin, and surfactin LP families from strains GB03, FZB42, and BSn5, with varying intensity. Using plate cultures, bacillomycin D fractions were detected in higher abundance in strains GB03, FZB42, and BSn5 in the presence of Fos. Additionally, the presence of Fos in dual plate culture triggered an increase in bacillomycin D production from the Bacillus strains. The study demonstrated the potent antagonistic effect of certain Bacillus strains (i.e., GB03, FZB42, BSn5) on Fos development. Our findings emphasize the crucial role of microbial interactions in shaping the co-existence of microbial assemblages.

Unpredictable soil conditions can affect the prevalence of a microbial symbiosis.

The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been widely demonstrated. We test for these associations using Dictyostelium discoideum social amoebae and their bacterial endosymbionts. D. discoideum commonly hosts endosymbiotic bacteria from three taxa: Paraburkholderia, Amoebophilus and Chlamydiae. Three species of facultative Paraburkholderia endosymbionts are the best studied and give hosts the ability to carry prey bacteria through the dispersal stage to new environments. Amoebophilus and Chlamydiae are obligate endosymbiont lineages with no measurable impact on host fitness. We tested whether the frequency of both single infections and coinfections of these symbionts were associated with the unpredictability of their soil environments by using symbiont presence-absence data from D. discoideum isolates from 21 locations across the eastern United States. We found that symbiosis across all infection types, symbiosis with Amoebophilus and Chlamydiae obligate endosymbionts, and symbiosis involving coinfections were not associated with any of our measures. However, unpredictable precipitation was associated with symbiosis in two species of Paraburkholderia, suggesting a link between unpredictable conditions and symbiosis.

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.

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.

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.

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.

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.

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.

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.

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.

Control of Pierce's Disease of Grape with Paraburkholderia phytofirmans PsJN in the Field.

Replicated field studies were conducted to evaluate the factors that could influence the efficacy of Paraburkholderia phytofirmans PsJN for the control of Pierce's disease of grape, as well as to determine the extent to which disease control was systemic within plants. Topical applications of PsJN with an organosilicon surfactant was an effective way to introduce this bacterium under field conditions and provided similar levels of disease control as its mechanical inoculation. Disease incidence in inoculated shoots was often reduced two- to threefold when PsJN was inoculated a single time as much as 3 weeks before Xylella fastidiosa and up to 5 weeks after the pathogen. Inoculation of a shoot with PsJN greatly decreased the probability of any symptoms rather than reducing the severity of disease, suggesting a systemic protective response of individual shoots. Although the likelihood of disease symptoms on shoots inoculated with the pathogen on PsJN-treated plants was lower than on control plants inoculated only with the pathogen, the protection conferred by PsJN was not experienced by all shoots on a given plant. This suggested that any systemic resistance was spatially limited. Whereas the population size of PsJN increased to more than 106 cells/g and spread more than 1 m within 12 weeks after its inoculation alone into grape, its population size subsequently decreased greatly after about 5 weeks, and its distal dispersal in stems was restricted when co-inoculated with X. fastidiosa. PsJN may experience collateral damage from apparent host responses induced when both species are present.

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.

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.

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.

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.