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.

Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple ß-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and ß-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

Cadmium biosorption and mechanism investigation using two cadmium-tolerant microorganisms isolated from rhizosphere soil of rice.

Microbial remediation of cadmium-contaminated soil offers advantages like environmental friendliness, cost-effectiveness, and simple operation. However, the efficacy of this remediation process relies on obtaining dominant strains and a comprehensive understanding of their Cd adsorption mechanisms. This study identified two Cd-resistant bacteria, Burkholderia sp. 1-22 and Bacillus sp. 6-6, with significant growth-promoting effects from rice rhizosphere soil. The strains showed remarkable Cd resistance up to ∼200 mg/L and alleviated Cd toxicity by regulating pH and facilitating bacterial adsorption of Cd. FTIR analysis showed crucial surface functional groups, like carboxyl and amino groups, on bacteria played significant roles in Cd adsorption. The strains could induce CdCO3 formation via a microbially induced calcium precipitation (MICP) mechanism, confirmed by SEM-EDS, X-ray analysis, and elemental mapping. Pot experiments showed these strains significantly increased organic matter and enzyme activity (e.g., urease, sucrase, peroxidase) in the rhizosphere soil versus the control group. These changes are crucial for restricting Cd mobility. Furthermore, strains 6-6 and 1-22 significantly enhance plant root detoxification of Cd, alleviating toxicity. Notably, increased pH likely plays a vital role in enhancing Cd precipitation and adsorption by strains, converting free Cd into non-bioavailable forms.

AHL-Based Quorum Sensing Regulates the Biosynthesis of a Variety of Bioactive Molecules in Bacteria.

Bacteria are social microorganisms that use communication systems known as quorum sensing (QS) to regulate diverse cellular behaviors including the production of various secreted molecules. Bacterial secondary metabolites are widely studied for their bioactivities including antibiotic, antifungal, antiparasitic, and cytotoxic compounds. Besides playing a crucial role in natural bacterial niches and intermicrobial competition by targeting neighboring organisms and conferring survival advantages to the producer, these bioactive molecules may be of prime interest to develop new antimicrobials or anticancer therapies. This review focuses on bioactive compounds produced under acyl homoserine lactone-based QS regulation by Gram-negative bacteria that are pathogenic to humans and animals, including the Burkholderia, Serratia, Pseudomonas, Chromobacterium, and Pseudoalteromonas genera. The synthesis, regulation, chemical nature, biocidal effects, and potential applications of these identified toxic molecules are presented and discussed in light of their role in microbial interactions.

Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

Genetic dissection of the degradation pathways for the mycotoxin fusaric acid in Burkholderia ambifaria T16.

IMPORTANCE: Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium.

Microscopy and modelling investigations on the morphology of the biofilm exopolysaccharide produced by Burkholderia multivorans strain C1576.

Bacteria form very often biofilms where they embed in a self-synthesized matrix exhibiting a gel-like appearance. Matrices offer several advantages, including defence against external threats and the easiness of intercellular communication. In infections, biofilm formation enhances bacteria resistance against antimicrobials, causing serious clinical problems for patients' treatments. Biofilm matrices are composed of proteins, extracellular DNA, and polysaccharides, the latter being the major responsible for matrix architecture. The repeating unit of the biofilm polysaccharide synthesized by Burkholderia multivorans strain C1576 contains two mannoses and two sequentially linked rhamnoses, one of them 50 % methylated on C-3. Rhamnose, a 6-deoxysugar, has lower polarity than other common monosaccharides and its methylation further reduces polarity. This suggests a possible role of this polysaccharide in the biofilm matrix; in fact, computer modelling and atomic force microscopy studies evidenced intra- and inter-molecular non-polar interactions both within polysaccharides and with aliphatic molecules. In this paper, the polysaccharide three-dimensional morphology was investigated using atomic force microscopy in both solid and solution states. Independent evidence of the polymer conformation was obtained by transmission electron microscopy which confirmed the formation of globular compact structures. Finally, data from computer dynamic simulations were used to model the three-dimensional structure.

The potential of Burkholderia thailandensis E264 for co-valorization of C5 and C6 sugars into multiple value-added bio-products.

Despite known metabolic versatility of Burkholderia spp., sugar metabolism and end-product synthesis patterns in Burkholderia thailandensis have been poorly characterized. This work has demonstrated that B. thailandensis is capable of simultaneously uptaking glucose and xylose and accumulating up to 64% of its dry mass as poly(3-hydroxyalkanoate) (PHA) biopolymers, resulting in a PHA titer of up to 3.8 g L-1 in shake flasks. Rhamnolipids - mainly (68-75%) in the form of Rha-Rha-C14-C14 - were produced concomitantly with a titer typically in the range of 0.2-0.4 g L-1. Gluconic and xylonic acids were also detected in titers of up to 5.3 g L-1, and while gluconic acid appeared to be back consumed, xylonic acid formed as a major end product. This first example of co-production of three products from mixed sugars using B. thailandensis paves the way for improving biorefinery economics.

Characterization of a cadmium-resistant functional bacteria (Burkholderia sp. SRB-1) and mechanism analysis at physiochemical and genetic level.

In this study, the capacity of cadmium (Cd)-resistant plant growth-promoting bacteria (PGPB) Burkholderia sp. SRB-1 (SRB-1) and its mechanisms were explored through morphological characterizations, biochemical response, plant growth-promoting traits, and functional gene expression patterns. The results showed that SRB-1 was an excellent Cd-resistant bacteria (MIC was 420 mg L-1), and its maximum Cd removal rate reached 72.25%. Biosorption was the main removal method of Cd for SRB-1, preventing intracellular Cd accumulation and maintaining cellular metabolism. Various functional groups on the cell wall were involved in Cd binding, which deposited as CdS and CdCO3 on the cell surface according to XPS analysis and might be critical for reducing Cd physiochemical toxicity. Furthermore, metals exporting (zntA, czcA, czcB, czcC), detoxification (dsbA, cysM), and antioxidation (katE, katG, SOD1) related genes were annotated in the SRB-1 genome. The results of Cd distribution and antioxidative enzyme activity in SRB-1 also illustrated that Cd2+ efflux and antioxidative response were the main intracellular Cd-resistant mechanisms. These conclusions were further verified by qRT-PCR analysis. Overall, the strategies of extracellular biosorption, cation efflux, and intracellular detoxification jointly build the Cd-resistant system, which invested Burkholderia sp. SRB-1 with potential for bioremediation in heavily Cd-contaminated environmental sites.

Understanding Autologous Spliceostatin Transcriptional Regulation to Derive Parts for Heterologous Expression in a Burkholderia Bacterial Host.

Burkholderia ß-Proteobacteria are emerging sources of natural products. We are interested in developing Burkholderia sp. FERM BP-3421 into a synthetic biology chassis to facilitate natural product discovery. FERM BP-3421 produces autologous spliceostatins on gram per liter scale. We reasoned that transcription factors and promoters that regulate spliceostatin biosynthesis would provide valuable parts for heterologous expression. Herein we demonstrate that fr9A encodes a pathway-specific transcriptional activator of spliceostatin biosynthesis. In-frame deletion of fr9A abolished spliceostatin production, which was restored by genetic complementation. Using transcriptomics and green fluorescent protein (GFP) reporter assays, we identified four fr9 promoters, three of which are activated by LuxR-type regulator Fr9A. We then constructed an Fr9A-regulated promoter system that was compared to benchmarks and effectively applied for GFP and capistruin lasso peptide expression in an optimized host background. Our findings enrich the genetic toolbox for optimizing heterologous expression and promoting the discovery and development of natural products from Burkholderia bacteria.

Genomic features and virulence characteristics of a rare Burkholderia thailandensis strain causing human infection.

Introduction. Burkholderia thailandensis is a clinically rare opportunistic pathogen in the genus Burkholderia, and the genomic features and virulence characteristics of B. thailandensis strains that cause human infection remain unclear.Gap Statement. B. thailandensis strains with different virulence induce different host innate immune responses in vitro.Aim. This work aimed to understand the sequence diversity, phylogenetic relationship, and virulence of B. thailandensis BPM causing human infection.Methodology. The comparative molecular and genomic analyses, and mouse infection studies were applied to analyse the virulence and genomic features of B. thailandensis BPM originating from China.Results. The whole genome sequence analysis showed that the genomes of BPM and other avirulent B. thailandensis strains were broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands. By examining species-specific genomic regions, we obtained molecular explanations for previously known differences in virulence and discovered the potential specific virulence-associated genes of BPM, which likely work together to confer the virulence of BPM. Significantly reduced LD50 and survival rates during mouse infection experiments were found in BPM compared to the avirulent B. thailandensis E264 (BtE264).Conclusion. Taken together, the results of this study provide basic information on the genomic features and virulence characteristics of the virulent B. thailandensis strain BPM, which is helpful for understanding its evolution as it relates to pathogenesis and environmental adaptability.

Sustainable rhamnolipids production in the next decade - Advancing with Burkholderia thailandensis as a potent biocatalytic strain.

Rhamnolipids are one of the most promising eco-friendly green glycolipids for bio-replacements of commercially available fossil fuel-based surfactants. However, the current industrial biotechnology practices cannot meet the required standards due to the low production yields, expensive biomass feedstocks, complicated processing, and opportunistic pathogenic nature of the conventional rhamnolipid producer strains. To overcome these problems, it has become important to realize non-pathogenic producer substitutes and high-yielding strategies supporting biomass-based production. We hereby review the inherent characteristics of Burkholderia thailandensis E264 which favor its competence towards such sustainable rhamnolipid biosynthesis. The underlying biosynthetic networks of this species have unveiled unique substrate specificity, carbon flux control and rhamnolipid congener profile. Acknowledging such desirable traits, the present review provides critical insights towards metabolism, regulation, upscaling, and applications of B. thailandensis rhamnolipids. Identification of their unique and naturally inducible physiology has proved to be beneficial for achieving previously unmet redox balance and metabolic flux requirements in rhamnolipids production. These developments in part are targeted by the strategic optimization of B. thailandensis valorizing low-cost substrates ranging from agro-industrial byproducts to next generation (waste) fractions. Accordingly, safer bioconversions can propel the industrial rhamnolipids in advanced biorefinery domains to promote circular economy, reduce carbon footprint and increased applicability as both social and environment friendly bioproducts.

Fast degradation of macro alkanes through activating indigenous bacteria using biosurfactants produced by Burkholderia sp.

Soil bacteria that produce biosurfactants can use total petroleum hydrocarbons (TPHs) as a carbon source. This study demonstrated that biosurfactants produced by Burkholderia sp. enhanced the recovery and synergism of soil microbial community, resulting in fast degradation of macro alkanes. Experiments were carried out by applying bio-stimulation after pre-oxidation to investigate the effects of nutrient addition on biosurfactant production, TPH degradation, and microbial community succession in the soil. The results presented that bio-stimulation could produce biosurfactants in high C/N (32.6) and C/H (13.3) conversion after pre-oxidation and increased the total removal rate of TPH (10.59-46.71%). The number of total bacteria had a rapid increase trend (2.94-8.50 Log CFU/g soil). The degradation rates of macro alkanes showed a 4.0-fold (48.07 mg/kg·d-1 versus 186.48 mg/kg·d-1) increase, and the bioremediation time of degrading macro alkanes saved 166 days. Further characterization revealed that the biosurfactants produced by Burkholderia sp. could activate indigenous bacteria to degrade macro alkanes rapidly. A shift in phylum from Actinomycetes to Proteobacteria was observed during bioremediation. The average relative abundance of the microbial community increased from 36.24 to 64.96%, and the predominant genus tended to convert from Allorhizobium (8.57%) to Burkholderia (15.95%) and Bacillus (15.70%). The co-occurrence network and Pearson correlation analysis suggested that the synergism of microbial community was the main reason for the fast degradation of macro alkanes in petroleum-contaminated soils. Overall, this study indicated the potential of the biosurfactants to activate and enhance the recovery of indigenous bacteria after pre-oxidation, which was an effective method to remediate petroleum-contaminated soils.

CRISPRi-Mediated Silencing of Burkholderia O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts.

The process of O-linked protein glycosylation is highly conserved across the Burkholderia genus and mediated by the oligosaccharyltransferase PglL. While our understanding of Burkholderia glycoproteomes has increased in recent years, little is known about how Burkholderia species respond to modulations in glycosylation. Utilizing CRISPR interference (CRISPRi), we explored the impact of silencing of O-linked glycosylation across four species of Burkholderia; Burkholderia cenocepacia K56-2, Burkholderia diffusa MSMB375, Burkholderia multivorans ATCC17616, and Burkholderia thailandensis E264. Proteomic and glycoproteomic analyses revealed that while CRISPRi enabled inducible silencing of PglL, this did not abolish glycosylation, nor recapitulate phenotypes such as proteome changes or alterations in motility that are associated with glycosylation null strains, despite inhibition of glycosylation by nearly 90%. Importantly, this work also demonstrated that CRISPRi induction with high levels of rhamnose leads to extensive impacts on the Burkholderia proteomes, which without appropriate controls mask the impacts specifically driven by CRISPRi guides. Combined, this work revealed that while CRISPRi allows the modulation of O-linked glycosylation with reductions up to 90% at a phenotypic and proteome levels, Burkholderia appears to demonstrate a robust tolerance to fluctuations in glycosylation capacity.

Rapid Biodegradation of the Organophosphorus Insecticide Acephate by a Novel Strain Burkholderia sp. A11 and Its Impact on the Structure of the Indigenous Microbial Community.

The acephate-degrading microbes that are currently available are not optimal. In this study, Burkholderia sp. A11, an efficient degrader of acephate, presented an acephate-removal efficiency of 83.36% within 56 h (100 mg·L-1). The A11 strain has a broad substrate tolerance and presents a good removal effect in the concentration range 10-1600 mg·L-1. Six metabolites from the degradation of acephate were identified, among which the main products were methamidophos, acetamide, acetic acid, methanethiol, and dimethyl disulfide. The main degradation pathways involved include amide bond breaking and phosphate bond hydrolysis. Moreover, strain A11 successfully colonized and substantially accelerated acephate degradation in different soils, degrading over 90% of acephate (50-200 mg·kg-1) within 120 h. 16S rDNA sequencing results further confirmed that the strain A11 gradually occupied a dominant position in the soil microbial communities, causing slight changes in the diversity and composition of the indigenous soil microbial community structure.

Integrated biochemical and transcriptomic analysis reveals the effects of Burkholderia sp. SRB-1 on cadmium accumulating in Chrysopogon zizanioides L. under Cd stress.

Application of plant growth-promoting rhizobacteria plays a vital role in enhancing phytoremediation efficiency. In this study, multiple approaches were employed to investigate the underlying mechanisms of Burkholderia sp. SRB-1 (SRB-1) on elevating Cd uptake and accumulation. Inoculation experiment indicated that SRB-1 could facilitate plant growth and Cd tolerance, as evidenced by the enhanced plant biomass and antioxidative enzymes activities. Cd content in plant shoots and roots increased about 36.56%-39.66% and 25.97%-130.47% assisted with SRB-1 when compared with control. Transcriptomics analysis revealed that SRB-1 upregulated expression of amiE, AAO1-2 and GA2-ox related to auxin and gibberellin biosynthesis in roots. Auxin and gibberellin, as hormone signals, regulated plant Cd tolerance and growth through activating hormone signal transduction pathways, which might also contribute to 67.94% increase of dry weight. The higher expression levels of ATP-binding cassette transporter subfamilies (ABCB, ABCC, ABCD and ABCG) in Chrysopogon zizanioides roots contributed to higher Cd uptake in Cd15 B (323.83 mg kg-1) than Cd15 (136.28 mg kg-1). Further, SRB-1 facilitated Cd migration from roots to shoots via upregulating the expression of Nramp, ZIP and HMA families. Our integrative analysis provided a molecular-scale perspective on Burkholderia sp. SRB-1 contributing to C. zizanioides performance.

Potential of Burkholderia sp. IMCC1007 as a biodetoxification agent in mycotoxin biotransformation evaluated by mass spectrometry and phytotoxicity analysis.

Microbial degradation is considered as an attractive method to eliminate exposure to mycotoxin that cause a serious threat in agriculture global industry and severe human health problems. Compared with other more prominent mycotoxin compounds, fusaric acid (FA) biodegradation has not been widely investigated. In this study, a fusaric acid-degrading bacterium Burkholderia sp. IMCC1007 was identified by 16 S rRNA gene sequencing and its detoxification characteristics were evaluated. This strain able to utilize FA as sole energy and carbon source with growth rate (µ) of 0.18 h- 1. Approximately 93% from the initial substrate FA concentration was almost degraded to the residual about 4.87 mg L- 1 after 12 h of incubation. The optimal degradation conditions for pH and temperature were recorded at 6.0 with 30 °C respectively. An efficient FA degradation of strain IMCC1007 suggested its potential significance to detoxification development. Accroding to LC-MS/Q-TOF analysis, FA was bio-transformed to 4-hydroxybenzoic acid (C7H6O3) and other possible metabolites. Plant treated with detoxified FA products exhibited reduction of wilting index, mitigating against FA phytoxicity effect on plant growth and photosynthesis activity. Phytotoxicity bioassay suggested that degradation product of IMCC1007 was not a potent harmful compound towards plants as compared to the parent compound, FA. As a conslusion, our study provides a new insight into the practical application of biodetoxifcation agent in controlling mycotoxin contamination.

Characterization of hydantoin-5-propionic acid amidohydrolase involved in ergothioneine utilization in Burkholderia sp. HME13.

In our previous study, ertABC genes encoding ergothionase, thiourocanate hydratase, and 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase were identified, all of which may be involved in ergothioneine utilization of Burkholderia sp. HME13. In this study, we identify the ertD gene encoding metal-dependent hydantoin-5-propionic acid amidohydrolase in this strain. Mn2+-containing ErtD showed maximum activity at 45 °C and pH 8.5 and was stable at temperatures up to 45 °C. The Km and Vmax values of Mn2+-containing ErtD for hydantoin-5-propionic acid were 2.8 m m and 16 U/mg, respectively. Real-time polymerase chain reaction (PCR) revealed that ertD expression levels in Burkholderia sp. HME13 cells cultivated in ergothioneine medium were 3.3-fold higher than those in cells cultivated in Luria-Bertani (LB) medium. ErtD activity in the crude extract from Burkholderia sp. HME13 cells cultured in ergothioneine medium was 0.018 U/mg, whereas that in LB medium was not detected. Accordingly, we suggest that ErtD is involved in ergothioneine utilization in this strain.

The Rhizopus Holobiont: A Model to Decipher Fungal-Bacterial-Viral Symbioses.

Rhizopus microsporus is an early-diverging fungal species that inhabits the soil, is used for the fermentation of diverse Asian and African foods, and can be a pathogen of plants, animals, and humans.Toxin-producing strains of R. microsporus live in symbiosis with Gram-negative betaproteobacteria from the genus Mycetohabitans (Burkholderia sensu lato). These bacterial endosymbionts increase the metabolic plasticity of the fungal holobiont by producing the "mycotoxins," control their asexual reproduction, and influence their sexual success. Recently, we identified two viruses of the genus Narnavirus in some R. microsporus strains that harbor Mycetohabitans. By eliminating bacteria and/or viruses from host R. microsporus strains, we have been able to study the role of these symbionts in fungal biology. Remarkably, the absence of these bacterial and viral symbionts decreases sexual reproduction. In this chapter, the method developed to eliminate and genotype the Narnavirus RmNV-20S and RmNV-23S in R. microsporus is described in detail.

Burkholderia gut symbiont induces insect host fecundity by modulating Kr-h1 gene expression.

Full-length cDNAs of the Broad-Complex (BR-C) from Riptortus pedestris were cloned. Moreover, Kr-h1 and BR-C expression levels in apo-symbiotic and symbiotic host insects were compared to verify whether they are modulated by Burkholderia gut symbionts. Interestingly, Kr-h1 expression level was significantly increased in symbiotic females. To determine how Kr-h1 affects fecundity in insects, the biosynthesis of two reproduction-associated proteins, hexamerin-α and vitellogenin, was investigated in R. pedestris females. Hexamerin-α and vitellogenin expression at the transcriptional and translational levels decreased in Kr-h1-suppressed symbiotic females, subsequently reduced egg production. These results suggest that Burkholderia gut symbiont modulates Kr-h1 expression to enhance ovarian development and egg production of R. pedestris by increasing the biosynthesis of the two proteins.