Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.

Gwanakosides A and B, 6-Deoxy-α-l-talopyranose-Bearing Aromatic Metabolites from a Streptomyces sp. and Coculture with Pandoraea sp.

Single-strain cultivation of a mountain soil-derived Streptomyces sp. GA02 and its coculture with Pandoraea sp. GA02N produced two aromatic products, gwanakosides A and B (1 and 2, respectively). Their spectroscopic analysis revealed that 1 is a new dichlorinated naphthalene glycoside and 2 is a pentacyclic aromatic glycoside. The assignment of the two chlorine atoms in 1 was confirmed by the analysis of its band-selective CLIP-HSQMBC spectrum. The sugars in the gwanakosides were identified as 6-deoxy-α-l-talopyranose based on 1H-1H coupling constants, Rotating frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, and chemical derivatization followed by spectroscopic and chromatographic analyses. The absolute configuration of 2, whose production was enhanced approximately 100-fold in coculture, was proposed based on a quantum mechanics-based chemical shift analysis method, DP4 calculations, and the chemically determined configuration of 6-deoxy-α-l-talopyranose. Gwanakoside A displayed inhibitory activity against pathogenic bacteria, including Staphylococcus aureus (MIC = 8 µg/mL) and Mycobacterium tuberculosis (MIC50 = 15 µg/mL), and antiproliferative activity against several human cancer cell lines (IC50 = 5.6-19.4 µM).

Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation.

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants.

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.

Metagenome-assembled genome of a Chitinophaga sp. and its potential in plant biomass degradation, as well of affiliated Pandoraea and Labrys species.

The prospection of new degrading enzymes of the plant cell wall has been the subject of many studies and is fundamental for industries, due to the great biotechnological importance of achieving a more efficient depolymerization conversion from plant polysaccharides to fermentable sugars, which are useful not only for biofuel production but also for various bioproducts. Thus, we explored the shotgun metagenome data of a bacterial community (CB10) isolated from sugarcane bagasse and recovered three metagenome-assembled genomes (MAGs). The genomic distance analyses, along with phylogenetic analysis, revealed the presence of a putative novel Chitinophaga species, a Pandoraea nosoerga, and Labrys sp. isolate. The isolation process for each one of these bacterial lineages from the community was carried out in order to relate them with the MAGs. The recovered draft genomes have reasonable completeness (72.67-100%) and contamination (0.26-2.66%) considering the respective marker lineage for Chitinophaga (Bacteroidetes), Pandoraea (Burkholderiales), and Labrys (Rhizobiales). The in-vitro assay detected cellulolytic activity (endoglucanases) only for the isolate Chitinophaga, and its genome analysis revealed 319 CAZymes, of which 115 are classified as plant cell wall degrading enzymes, which can act in fractions of hemicellulose and pectin. Our study highlights the potential of this Chitinophaga isolate provides several plant-polysaccharide-degrading enzymes.

Genomic Aromatic Compound Degradation Potential of Novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov.

We performed a taxonomic and comparative genomics analysis of 67 novel Paraburkholderia isolates from forest soil. Phylogenetic analysis of the recA gene revealed that these isolates formed a coherent lineage within the genus Paraburkholderia that also included Paraburkholderiaaspalathi, Paraburkholderiamadseniana, Paraburkholderiasediminicola, Paraburkholderiacaffeinilytica, Paraburkholderiasolitsugae and Paraburkholderiaelongata and four unidentified soil isolates from earlier studies. A phylogenomic analysis, along with orthoANIu and digital DNA-DNA hybridization calculations revealed that they represented four different species including three novel species and P. aspalathi. Functional genome annotation of the strains revealed several pathways for aromatic compound degradation and the presence of mono- and dioxygenases involved in the degradation of the lignin-derived compounds ferulic acid and p-coumaric acid. This co-occurrence of multiple Paraburkholderia strains and species with the capacity to degrade aromatic compounds in pristine forest soil is likely caused by the abundant presence of aromatic compounds in decomposing plant litter and may highlight a diversity in micro-habitats or be indicative of synergistic relationships. We propose to classify the isolates representing novel species as Paraburkholderia domus with LMG 31832T (=CECT 30334) as the type strain, Paraburkholderia nemoris with LMG 31836T (=CECT 30335) as the type strain and Paraburkholderia haematera with LMG 31837T (=CECT 30336) as the type strain and provide an emended description of Paraburkholderia sediminicola Lim et al. 2008.

Proteomics, Lipidomics, Metabolomics, and 16S DNA Sequencing of Dental Plaque From Patients With Diabetes and Periodontal Disease.

Oral microbiome influences human health, specifically prediabetes and type 2 diabetes (Pre-DM/DM) and periodontal diseases (PDs), through complex microbial interactions. To explore these relations, we performed 16S rDNA sequencing, metabolomics, lipidomics, and proteomics analyses on supragingival dental plaque collected from individuals with Pre-DM/DM (n = 39), Pre-DM/DM and PD (n = 37), PD alone (n = 11), or neither (n = 10). We identified on average 2790 operational taxonomic units and 2025 microbial and host proteins per sample and quantified 110 metabolites and 415 lipids. Plaque samples from Pre-DM/DM patients contained higher abundance of Fusobacterium and Tannerella than plaques from metabolically healthy patients. Phosphatidylcholines, plasmenyl phosphatidylcholines, ceramides containing non-OH fatty acids, and host proteins related to actin filament rearrangement were elevated in plaques from PD versus non-PD samples. Cross-omic correlation analysis enabled the detection of a strong association between Lautropia and monomethyl phosphatidylethanolamine (PE-NMe), which is striking because synthesis of PE-NMe is uncommon in oral bacteria. Lipidomics analysis of in vitro cultures of Lautropia mirabilis confirmed the synthesis of PE-NMe by the bacteria. This comprehensive analysis revealed a novel microbial metabolic pathway and significant associations of host-derived proteins with PD.

Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria.

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

2,5-Furandicarboxylic acid production from furfural by sequential biocatalytic reactions.

2,5-Furandicarboxylic acid (FDCA) is a valuable compound that can be synthesized from biomass-derived hydroxymethylfurfural (HMF), and holds great potential as a promising replacement for petroleum-based terephthalic acid in the production of polyamides, polyesters, and polyurethanes used universally. However, an economical large-scale production strategy for HMF from lignocellulosic biomass is yet to be established. This study aimed to design a synthetic pathway that can yield FDCA from furfural, whose industrial production from lignocellulosic biomass has already been established. This artificial pathway consists of an oxidase and a prenylated flavin mononucleotide (prFMN)-dependent reversible decarboxylase, catalyzing furfural oxidation and carboxylation of 2-furoic acid, respectively. The prFMN-dependent reversible decarboxylase was identified in an isolated strain, Paraburkholderia fungorum KK1, whereas an HMF oxidase from Methylovorus sp. MP688 exhibited furfural oxidation activity and was used as a furfural oxidase. Using Escherichia coli cells coexpressing these proteins, as well as a flavin prenyltransferase, FDCA could be produced from furfural via 2-furoic acid in one pot.

Linking prokaryotic community composition to carbon biogeochemical cycling across a tropical peat dome in Sarawak, Malaysia.

Tropical peat swamp forest is a global store of carbon in a water-saturated, anoxic and acidic environment. This ecosystem holds diverse prokaryotic communities that play a major role in nutrient cycling. A study was conducted in which a total of 24 peat soil samples were collected in three forest types in a tropical peat dome in Sarawak, Malaysia namely, Mixed Peat Swamp (MPS), Alan Batu (ABt), and Alan Bunga (ABg) forests to profile the soil prokaryotic communities through meta 16S amplicon analysis using Illumina Miseq. Results showed these ecosystems were dominated by anaerobes and fermenters such as Acidobacteria, Proteobacteria, Actinobacteria and Firmicutes that cover 80-90% of the total prokaryotic abundance. Overall, the microbial community composition was different amongst forest types and depths. Additionally, this study highlighted the prokaryotic communities' composition in MPS was driven by higher humification level and lower pH whereas in ABt and ABg, the less acidic condition and higher organic matter content were the main factors. It was also observed that prokaryotic diversity and abundance were higher in the more oligotrophic ABt and ABg forest despite the constantly waterlogged condition. In MPS, the methanotroph Methylovirgula ligni was found to be the major species in this forest type that utilize methane (CH4), which could potentially be the contributing factor to the low CH4 gas emissions. Aquitalea magnusonii and Paraburkholderia oxyphila, which can degrade aromatic compounds, were the major species in ABt and ABg forests respectively. This information can be advantageous for future study in understanding the underlying mechanisms of environmental-driven alterations in soil microbial communities and its potential implications on biogeochemical processes in relation to peatland management.

Impact of root-associated strains of three Paraburkholderia species on primary and secondary metabolism of Brassica oleracea.

Several root-colonizing bacterial species can simultaneously promote plant growth and induce systemic resistance. How these rhizobacteria modulate plant metabolism to accommodate the carbon and energy demand from these two competing processes is largely unknown. Here, we show that strains of three Paraburkholderia species, P. graminis PHS1 (Pbg), P. hospita mHSR1 (Pbh), and P. terricola mHS1 (Pbt), upon colonization of the roots of two Broccoli cultivars led to cultivar-dependent increases in biomass, changes in primary and secondary metabolism and induced resistance against the bacterial leaf pathogen Xanthomonas campestris. Strains that promoted growth led to greater accumulation of soluble sugars in the shoot and particularly fructose levels showed an increase of up to 280-fold relative to the non-treated control plants. Similarly, a number of secondary metabolites constituting chemical and structural defense, including flavonoids, hydroxycinnamates, stilbenoids, coumarins and lignins, showed greater accumulation while other resource-competing metabolite pathways were depleted. High soluble sugar generation, efficient sugar utilization, and suppression or remobilization of resource-competing metabolites potentially contributed to curb the tradeoff between the carbon and energy demanding processes induced by Paraburkholderia-Broccoli interaction. Collectively, our results provide a comprehensive and integrated view of the temporal changes in plant metabolome associated with rhizobacteria-mediated plant growth promotion and induced resistance.

Phenazine-1-carboxylic acid (PCA) produced by Paraburkholderia phenazinium CK-PC1 aids postgermination growth of Xyris complanata seedlings with germination induced by Penicillium rolfsii Y-1.

Symbiosis of Penicillium rolfsii Y-1 is essential for the seed germination of Hawaii yellow-eyed grass (Xyris complanata). However, the local soil where the plants grow naturally often suppresses the radicle growth of the seedlings. This radicle growth was drastically restored by coinoculation of Paraburkholderia phenazinium isolate CK-PC1, which is a rhizobacterium of X. complanata. It was found that the isolate CK-PC1 produced phenazine-1-carboxylic acid (PCA, 1) as a major metabolite. The biological effects of PCA (1) were investigated using the seeds of X. complanata and Mung bean (Vigna radiata) and it was uncovered that the symbiosis of the isolate CK-PC1was essential for the postgermination growth of X. complanata and the metabolite PCA (1) might partially contribute to promote the growth of the plants.

A chronic strain of the cystic fibrosis pathogen Pandoraea pulmonicola expresses a heterogenous hypo-acylated lipid A.

Pandoraea sp. is an emerging Gram-negative pathogen in cystic fibrosis causing severe and persistent inflammation and damage of the lungs. The molecular mechanisms underlying the high pathogenicity of Pandoraea species are still largely unknown. As Gram-negatives, Pandoraea sp. express lipopolysaccharides (LPS) whose recognition by the host immune system triggers an inflammatory response aimed at the bacterial eradication from the infected tissues. The degree of the inflammatory response strongly relies on the fine structure of the LPS and, in particular, of its glycolipid moiety, i.e. the lipid A. Here we report the structure of the lipid A isolated from the LPS of a chronic strain of P. pulmonicola (RL 8228), one of the most virulent identified so far among the Pandoraea species. Our data demonstrated that the examined chronic strain produces a smooth-type LPS with a complex mixture of hypoacylated lipid A species displaying, among other uncommon characteristics, the 2-hydroxylation of some of the acyl chains and the substitution by an additional glucosamine on one or both the phosphate groups.

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

Dual transcriptomics and proteomics analyses of the early stage of interaction between Caballeronia mineralivorans PML1(12) and mineral.

Minerals and rocks represent essential reservoirs of nutritive elements for the long-lasting functioning of forest ecosystems developed on nutrient-poor soils. While the presence of effective mineral weathering bacteria was evidenced in the rhizosphere of different plants, the molecular mechanisms involved remain uncharacterized. To fill this gap, we combined transcriptomic, proteomics, geo-chemical and physiological analyses to decipher the potential molecular mechanisms explaining the mineral weathering effectiveness of strain PML1(12) of Caballeronia mineralivorans. Considering the early-stage of the interaction between mineral and bacteria, we identified the genes and proteins differentially expressed when: (i) the environment is depleted of certain essential nutrients (i.e., Mg and Fe), (ii) a mineral is added and (iii) the carbon source (i.e., glucose vs mannitol) differs. The integration of these data demonstrates that strain PML1(12) is capable of (i) mobilizing iron through the production of a non-ribosomal peptide synthetase-independent siderophore, (ii) inducing chemotaxis and motility in response to nutrient availability and (iii) strongly acidifying its environment in the presence of glucose using a suite of GMC oxidoreductases to weather mineral. These results provide new insights into the molecular mechanisms involved in mineral weathering and their regulation and highlight the complex sequence of events triggered by bacteria to weather minerals.

Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283.

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO), catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of a broader substrate range, the BphAES283M, BphAEp4-S283M, and BphAERR41-S283M variants were created from the parent enzymes BphAELB400, BphAEp4, and BphAERR41, respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km values of BphAES283M, BphAEp4-S283M, and BphAERR41-S283M were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-tetrachlorobiphenyl (2,3',4,4'-CB), 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-CB), and 2,3',4,4',5-pentachlorobiphenyl (2,3',4,4',5-CB). We compared the catalytic reactions of BphAELB400 and its variants toward 2,2'-dichlorobiphenyl (2,2'-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6-chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes.IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3',4,4'-CB, 2,2',6,6'-CB, and 2,3',4,4',5-CB. Meanwhile, the Ser283Met substitution altered the regiospecificity of BphAE toward 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3 to 6 chlorines), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

The roles of extracellular polymeric substances of Pandoraea sp. XY-2 in the removal of tetracycline.

In this study, the roles of extracellular polymeric substances (EPSs) excreted by Pandoraea sp. XY-2 in the removal of tetracycline (TC) were investigated. In the early stage, TC in the solution was mainly removed by the adsorption of EPSs, which accounted for 20% of TC. Thereafter, large amount of TC was transported into the intracellular and biodegraded. EPSs was extracted and the contents of polyprotein and polysaccharides reached their maximum values (30.84 mg/g and 11.15 mg/g) in the first four days. Fourier transform infrared spectroscopy analysis revealed that hydroxyl, methylidyne, methylene and amide I groups in EPSs participated in the adsorption of TC. Furthermore, three-dimensional excitation-emission matrix fluorescence spectroscopy analysis revealed that TC caused the quenching of EPSs fluorescent groups. The quenching mechanism was attributed to static quenching and protein-like substances in EPSs from Pandoraea sp. XY-2 dominated the TC adsorption process. Bioinformatic analysis of Pandoraea sp. XY-2 genome identified multiple genes involved in exopolysaccharide synthesis and EPSs formation. The insights gained in this study might provide a better understanding about the adsorption process of EPSs in tetracycline-contaminated environment.

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases.

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

The promiscuity of Phaseolus vulgaris L. (common bean) for nodulation with rhizobia: a review.

Phaseolus vulgaris L. (common bean) is a legume indigenous to American countries currently cultivated in all continents, which is nodulated by different rhizobial species and symbiovars. Most of species able to nodulate this legume worldwide belong to the genus Rhizobium, followed by those belonging to the genera Ensifer (formerly Sinorhizobium) and Pararhizobium (formerly Rhizobium) and minority by species of the genus Bradyrhizobium. All these genera belong to the phylum alpha-Proteobacteria, but the nodulation of P. vulgaris has also been reported for some species belonging to Paraburkholderia and Cupriavidus from the beta-Proteobacteria. Several species nodulating P. vulgaris were originally isolated from nodules of this legume in American countries and are linked to the symbiovars phaseoli and tropici, which are currently present in other continents probably because they were spread in their soils together with the P. vulgaris seeds. In addition, this legume can be nodulated by species and symbiovars originally isolated from nodules of other legumes due its high promiscuity, a concept currently related with the ability of a legume to be nodulated by several symbiovars rather than by several species. In this article we review the species and symbiovars able to nodulate P. vulgaris in different countries and continents and the challenges on the study of the P. vulgaris endosymbionts diversity in those countries where they have not been studied yet, that will allow to select highly effective rhizobial strains in order to guarantee the success of P. vulgaris inoculation.

Understanding the role of bacterial cellular adsorption, accumulation and bioavailability regulation by biosurfactant in affecting biodegradation efficacy of polybrominated diphenyl ethers.

Microbiological degradation is often considered as an important strategy to reduce the risks of polybrominated diphenyl ethers (PBDEs), which are environmentally widespread and harmful to human health and wildlife. With the well-identified aerobic bacteria, i.e. B. xenovorans LB400, the biodegradation of 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) occurred efficiently in conformity to the first-order kinetics and showed the strong dependence on initial concentration of pollutant and bioavailability regulation by biosurfactant. The mild increase of initial concentration of BDE-47 would enhance biodegradation whereas the excessive increase failed due to the oxidative stress or cytotoxicity to bacteria. Rather than the bacterial extracellular adsorption that was bioactively-mediated in thermodynamics, the intracellular accumulations at different time gradients showed the negative correlation with biodegradation efficiency of BDE-47. The spontaneous biodegradation of pollutant should be sourced from the gradual reduction of intracellular accumulation. Though the improved bioavailability of BDE-47 by sucrose fatty acid ester (SFAE) hardly altered the extracellular adsorption, the bacterial intracellular accumulation was indicated to increase continuously with used amount of biosurfactant and then decrease for the cellular morphological damage, and interestingly it appeared to be temporary reservoir for prompt delivery to biodegradation in light of the opposite variation tendency with time.