Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion.

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.

Microbial community structure and potential function associated with poly-3-hydroxybutyrate biopolymer-boosted activation of peroxymonosulfate for waste-activated sludge decontamination.

Excess waste-activated sludge (WAS) is a major biosolid management problem due to its biohazardous and recalcitrant content of phthalate esters (PAEs). This study aimed to assess the combined use of biopolymer, poly-3-hydroxybutyrate and peroxymonosulfate to degrade PAEs and decontaminate WAS. Poly-3-hydroxybutyrate was biosynthesized by Cupriavidus sp. L7L. The combined poly-3-hydroxybutyrate and peroxymonosulfate process removed 86 % of PAEs from WAS in 12 h. The carbonyl groups of poly-3-hydroxybutyrate were conducive to peroxymonosulfate activation leading to PAE degradation followed the radical pathway and surface-mediated electron transfer. Poly-3-hydroxybutyrate and peroxymonosulfate also enriched the PAE-biodegrading microbes in WAS. The microbial population and the functional composition in response to peroxymonosultate treatment was identified, with the genus Sulfurisoma being the most abundant. This synergistic treatment, i.e., advanced oxidation process, was augmented by highly promising microbial polyesters, exhibited important implications for WAS pretreatment toward circular bioeconomy that encompasses carbon-neutral biorefinery and mitigate pollution.

Metal-free catalysis for organic micropollutant degradation in waste activated sludge via poly(3-hydroxybutyrate) biopolymers using Cupriavidus sp. L7L coupled with peroxymonosulfate.

This study employed a novel and environment-friendly biopolymer/oxidant catalytic system, viz., poly(3-hydroxybutyrate)/peroxymonosulfate (PHB/PMS), for pretreating wastewater sludge for the first time. Under optimal conditions, i.e., 3.1 × 10-4 M of PMS and 3.3 g/L of PHB at pH = 6.0, the PAHs in the sludge matrix was decreased by 79 % in 12 h. Increase in salinity (75 % synthetic seawater) achieved 83 % of PAHs degradation. Functional groups (CO) of the biopolymer matrix were active centers for biopolymer-mediated electron transfer that produced reactive oxygen species (SO4-, HO, and 1O2) for adsorption and catalytic oxidation of PAHs in the sludge. Functional metagenomic analysis revealed the main genus, Conexibacter (phylum, Actinobacteria) exhibited PAH-degrading function with high efficiency in the biodegradation of PAHs from sludge pretreated with PHB/PMS. Coupling chemical oxidation and biostimulation using bacterial polymer-based biomaterials is effective and beneficial for pretreating wastewater sludge toward circular bioeconomy.

Rhodobacter amnigenus sp. nov. and Rhodobacter ruber sp. nov., isolated from freshwater habitats.

Two bacterial strains, designated HSP-20T and CCP-1T, isolated from freshwater habitats in Taiwan, were characterized by polyphasic taxonomy. Both strains were Gram-stain-negative, aerobic, non-motile and rod-shaped. Cells of strains HSP-20T and CCP-1T formed pink and dark red coloured colonies, respectively. Both strains contained bacteriochlorophyll a, and showed optimum growth under anaerobic conditions by photoheterotrophy, but no growth by photoautotrophy. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences indicated that both strains belonged to the genus Rhodobacter. Analysis of 16S rRNA gene sequences showed that strains HSP-20T and CCP-1T shared 98.3 % sequence similarity and were closely related to Rhodobacter tardus CYK-10T (96.0 %) and Rhodobacter flagellatus SYSU G03088T (96.0 %), respectively. Both strains shared common chemotaxonomic characteristics including Q-10 as the major isoprenoid quinone, C18 : 1 ω7c as the predominant fatty acid, and phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as the main polar lipids. The DNA G+C content of both strains was 66.2 mol%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between these two novel isolates and their closest relatives were below the cut-off values of 95-96, 90 and 70 %, respectively, used for species demarcation. On the basis of phenotypic and genotypic properties and phylogenetic inference, both strains should be classified as novel species within the genus Rhodobacter, for which the names Rhodobacter amnigenus sp. nov. (=BCRC 81193T=LMG 31334T) and Rhodobacter ruber sp. nov. (=BCRC 81189T=LMG 31335T) are proposed.

Zophobihabitans entericus gen. nov., sp. nov., a new member of the family Orbaceae isolated from the gut of a superworm Zophobas morio.

A novel bacterial strain, designated IPMB12T, isolated from the gut of the superworm Zophobas morio in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, facultatively anaerobic, non-motile, coccoid or rod-shaped and formed translucent colonies. Optimal growth occurred at 25-37 °C, pH 9-10, and with 0-2 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain IPMB12T is affiliated with genus in the the family Orbaceae in the class Gammaproteobacteria. Strain IPMB12T was most closely related to Gilliamella mensalis LMG 29880T with a 94.6 % 16S rRNA gene sequence similarity. Strain IPMB12T showed less than 71.6 % average nucleotide identity, less than 71.5 % average amino acid identity and less than 21.2 % digital DNA-DNA hybridization identity compared to the strains of related genera within the family Orbaceae. The major fatty acids of strain IPMB12T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and C14 : 0. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one uncharacterized phosphoaminoglycolipid and one uncharacterized aminophospholipid. The major isoprenoid quinone was Q-8. Genomic DNA G+C content of strain IPMB12T was 39.3 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain IPMB12T represents a novel species of a new genus in the family Orbaceae, for which the name Zophobihabitans entericus gen. nov., sp. nov. is proposed. The type strain is IPMB12T (=BCRC 80908T =LMG 32079T=KCTC 82347T=KACC 22323T).

The polyhydroxyalkanoate (PHA) synthase 1 of Pseudomonas sp. H9 synthesized a 3-hydroxybutyrate-dominant hybrid of short- and medium-chain-length PHA.

Pseudomonas sp. H9 (H9), an environmental strain isolated from a riverbank soil sample collected in southern Taiwan, is Gram-negative and shares a 99 % sequence identity to Pseudomonas putida KT2440 based on 16S rRNA gene analysis. H9 produced novel polyhydroxyalkanoates (PHA) including a hybrid PHA, comprised of 3-hydroxyvalerate (37 mol%) and medium-chain-length (MCL) monomers from valerate, as well as a 3-hydroxyhexanoate-dominant (93 mol%) MCL-PHA from hexanoate. Next-generation sequencing analysis showed H9 had a typical class II PHA operon, consisted of phaC1H9-phaZH9-phaC2H9, in which phaC1H9 was the sole active PHA synthase in H9. Deletion of phaC1H9 gene led to a complete loss of its PHA accumulation capability. Knockout of phaC2H9 gene, in contrast, affected neither bacterial growth nor PHA accumulation. When co-expressed with the phaAB genes of Ralstonia eutropha H16 in the PHA mutant strain Pseudomonas sp. H9ΔC1, phaC1H9 synthesized a hybrid PHA consisted of 3-hydroxybutyrate (3HB) (75 mol%) and MCL-monomers, confirmed in analyses using hot-acetone fractionation and 13C-NMR spectroscopy. As a novel PHA synthase, PhaC1H9 possesses a broad substrate specificity to synthesize a hybrid of SCL- and MCL-PHA, known to have many mechanical properties for potential applications.

Sphingomonas lacunae sp. nov., isolated from a freshwater pond.

A novel bacterial strain, designated CSW-10T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed yellow-coloured colonies. Optimal growth occurred at 30 °C, pH 7, and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain CSW-10T formed a phylogenetic lineage in the genus Sphingomonas. The 16S rRNA gene sequence similarity indicated that strain CSW-10T was most closely related to Sphingomonas fonticola TNR-2T (97.6%). Strain CSW-10T showed 69.8-70.7% average nucleotide identity and 19.0-23.0% digital DNA-DNA hybridization identity with the strains of other related Sphingomonas species. The major fatty acids of strain CSW-10T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C17:1 ω6c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, one uncharacterized sphingoglycolipid, five uncharacterized aminophospholipids, one uncharacterized phospholipid and one uncharacterized lipid. The predominant polyamines were homospermidine and spermidine. The major isoprenoid quinone was Q-10. Genomic DNA G+C content of strain CSW-10T was 62.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain CSW-10T should represent a novel species of the genus Sphingomonas, for which the name Sphingomonas lacunae sp. nov. is proposed. The type strain is CSW-10T (=BCRC 81190T =LMG 31340T).

Novosphingobium ovatum sp. nov., isolated from a freshwater mesocosm.

A bacterial strain, designated FSY-8T, was isolated from a freshwater mesocosm in Taiwan and characterized using the polyphasic taxonomy approach. Cells of strain FSY-8T were aerobic, Gram-stain-negative, rod-shaped, non-motile and formed yellow coloured colonies on Reasoner's 2A agar. Growth occurred at 20-40 °C (optimum, 30-37 °C) and pH 5-7 (optimum, pH 6) and in the presence of 0-0.5 % NaCl (optimum, 0 %, w/v). The major fatty acids (>10 %) of strain FSY-8T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipid, diphosphatidylglycerol, an uncharacterized aminophospholipid, an uncharacterized glycolipid and an uncharacterized lipid. The major polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content was 64.8 mol %. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain FSY-8T formed a phylogenetic lineage in the genus Novosphingobium. Strain FSY-8T showed 71.6-77.2 % average nucleotide identity and 19.9-22.8 % digital DNA-DNA hybridization identity with the strains of other Novosphingobium species. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain FSY-8T should be classified in a novel species of the genus Novosphingobium, for which the name Novosphingobium ovatum sp. nov. is proposed. The type strain is FSY-8T (=BCRC 81051T=LMG 30053T=KCTC 52812T).

Rhodovarius crocodyli sp. nov., isolated from a crocodile pond.

Bacterial strain CCP-6T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain CCP-6T is affiliated with species in the genus Rhodovarius. Strain CCP-6T was most closely related to Rhodovarius lipocyclicus CCUG 44693T with a 98.9% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed light pink-coloured colonies. Optimal growth occurred at 30 °C, pH 6 and in the absence of NaCl. The major fatty acids of strain CCP-6T were C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidyldimethylethanolamine, phosphatidylmethylethanolamine, diphosphatidylglycerol, three unidentified aminophospholipids and an unidentified phospholipid. The predominant polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content of the genomic DNA was 69.3 mol%. Strain CCP-6T showed 85.8% average nucleotide identity and 14.5% digital DNA-DNA hybridization identity with Rhodovarius lipocyclicus CCUG 44693T. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain CCP-6T represents a novel species in the genus Rhodovarius, for which the name Rhodovarius crocodyli sp. nov. is proposed. The type strain is CCP-6T (=BCRC 81095T=LMG 30310T=KCTC 62188T).

Ideonella livida sp. nov., isolated from a freshwater lake.

A novel bacterial strain, designated TBM-1T, isolated from a freshwater lake in Taiwan, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain TBM-1T formed a phylogenetic lineage in the genus Ideonella. Analysis of 16S rRNA gene sequences showed that strain TBM-1T was most closely related to Ideonella dechloratans CCUG 30898T with 98.4 % sequence similarity. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain TBM-1T and closely related strains of the genus Ideonella were 74.4-77.5 %, 69.7-75.4 % and 19.8-21.8 %, respectively, supporting that strain TBM-1T represents a novel species of the genus Ideonella. Cells were Gram-stain-negative, motile by means of a single polar flagellum, rod-shaped and formed blue colonies. Optimal growth occurred at 30 °C, pH 6 and 0 % NaCl. The predominant fatty acids of strain TBM-1T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two uncharacterized aminophospholipids and two uncharacterized phospholipids. The main polyamine was putrescine. The major isoprenoid quinone was Q-8. The estimated genome size was 5.26 Mb, with an average G+C content of 70.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain TBM-1T should be classified in a novel species of the genus Ideonella, for which the name Ideonella livida sp. nov. is proposed. The type strain is TBM-1T (=BCRC 81199T =LMG 31339T).

Cultivation temperature modulated the monomer composition and polymer properties of polyhydroxyalkanoate synthesized by Cupriavidus sp. L7L from levulinate as sole carbon source.

Cupriavidus sp. L7L, a newly isolated wild-type soil bacterium, was found to synthesize polyhydroxyalkanoate (PHA) terpolymers from levulinic acid (LA), a top bio-based platform chemical, as the sole carbon source. NMR spectra showed that the terpolymers consisted mainly of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and a small amount (2.1-4.8 mol%) of 4-hydroxyvalerate (4HV). The combination of monomers together with high molecular weights improved markedly the thermal and mechanical properties of the terpolymers. Increasing cultivation temperature of Cupriavidus sp. L7L increased the proportions of 3HB and decreased the proportions of 3HV. A fed-batch fermentation using LA as the sole carbon source without pH control produced a biomass of 15.8 dry weight g L-1 that contained 81 dry weight% of a terpolymer P(3HB-co-33.7 mol% 3HV-co-2.1 mol% 4HV), equaling a productivity of 0.213 g PHA L-1 h-1. The terpolymer showed a melting point of 92 °C and elongation at break of 630%, compared to 84.6 °C and 462%, respectively, for the copolymer P(3HB-co-35.9 mol% 3HV) that contained no 4HV. This study showed that Cupriavidus sp. L7L exhibited a great potential for producing PHA polymers with excellent mechanical property that could be modulated by cultivation temperature when cultivated exclusively in LA.

Litoribrevibacter euphylliae sp. nov., isolated from the torch coral Euphyllia glabrescens.

Strain Eup a-2T, isolated from the torch coral Euphyllia glabrescens, was characterized using a polyphasic taxonomy approach. Cells of strain Eup a-2T were Gram-negative, aerobic and motile by three polar flagella and formed translucent colonies. Optimal growth occurred at 25 °C, pH 6-8 and in the presence of 2-4 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Eup a-2T belonged to the genus Litoribrevibacter and showed the highest levels of sequence similarity with respect to Litoribrevibacter albus Y32T (97.8 %). Strain Eup a-2T contained summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0 as the predominant fatty acids. The predominant isoprenoid quinone was Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphophatidylglycerol. Genomic DNA G+C content of strain Eup a-2T was 49.1 mol%. The DNA-DNA hybridization value for strain Eup a-2T with L. albus Y32T was less than 30 %. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain Eup a-2T should be classified as a novel species of the genus Litoribrevibacter, for which the name Litoribrevibactereuphylliae sp. nov. is presented. The type strain is Eup a-2T (=BCRC 81004T=LMG 29725T=KCTC 52438T).

Thalassotalea coralli sp. nov., isolated from the torch coral Euphyllia glabrescens.

Strain Eup a-8T, isolated from a torch coral Euphyllia glabrescens, was characterized using a polyphasic taxonomy approach. Cells of strain Eup a-8T were Gram-staining-negative, aerobic, motile by means of a single polar flagellum, poly-ß-hydroxybutyrate-containing, rod-shaped and formed white colonies. Optimal growth occurred at 25-30 °C, pH 7-8, and in the presence of 2 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Eup a-8T belonged to the genus Thalassotalea and showed the highest levels of sequence similarity with respect to Thalassotalea ganghwensis JC2041T (97.1 %). Strain Eup a-8T contained C17 : 1ω8c, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C14 : 0 and iso-C16 : 0 as the predominant fatty acids. The only isoprenoid quinone was Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and one uncharacterized phospholipid. Genomic DNA G+C content of strain Eup a-8T was 41.5 mol%. The DNA-DNA hybridization value for strain Eup a-8T with Thalassotalea ganghwensis JC2041T was less than 70 %. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain Eup a-8T should be classified as a novel species of the genus Thalassotalea, for which the name Thalassotalea coralli sp. nov. is proposed. The type strain is Eup a-8T (=BCRC 80967T=LMG 29478T=KCTC 52169T).

Endozoicomonas acroporae sp. nov., isolated from Acropora coral.

Strain Acr-14T, isolated from Acropora coral, was characterized by using a polyphasic taxonomy approach. Cells of strain Acr-14T were Gram-stain-negative, aerobic, non-motile, poly-ß-hydroxybutyrate-accumulating, rod-shaped and formed creamy white colonies. Optimal growth occurred at 30 °C, pH 7 and in the presence of 2 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Acr-14T belonged to the genus Endozoicomonas and was most closely related to Endozoicomonas atrinae WP70T with sequence similarity of 96.7 %. Strain Acr-14T contained summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0 as the predominant fatty acids. The predominant isoprenoid quinone was Q-9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of strain Acr-14T was 49.1 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrated that strain Acr-14T should be classified as a novel species of the genus Endozoicomonas, for which the name Endozoicomonas acroporae sp. nov. is presented. The type strain is Acr-14T (=BCRC 80922T=LMG 29482T=KCTC 42901T).

Planctobacterium marinum gen. nov., sp. nov., a new member of the family Alteromonadaceae isolated from seawater.

A bacterial strain designated K7T was isolated from the South China Sea and characterized using a polyphasic taxonomic approach. Cells of strain K7T were Gram-stain-negative, aerobic, poly-ß-hydroxybutyrate-accumulating, motile by means of a monopolar flagellum, non-spore forming rods surrounded by a thick capsule and forming yellow colonies. Growth occurred at 4-35 °C (optimum, 25-30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and with 0.5-10 % (w/v) NaCl [optimum, 1-4 % (w/v)]. The predominant fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8 and the DNA G+C content was 46.5 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, one uncharacterized phospholipid, two uncharacterized aminophospholipids and five uncharacterized lipids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain K7T formed a distinct lineage with respect to closely related genera in the family Alteromonadaceae. Strain K7T was most closely related to Aestuariibacter, Aliiglaciecola, Paraglaciecola and Glaciecola, and the levels of 16S rRNA gene sequence similarity with respect to the type species of related genera were less than 95 %. On the basis of the genotypic and phenotypic data, strain K7T represents a novel species of a new genus of the family Alteromonadaceae, for which the name Planctobacterium marinum gen. nov., sp. nov. is proposed. The type strain of Planctobacterium marinum is K7T (=BCRC 80901T=LMG 28835T=KCTC 42657T).

Gemmobacter tilapiae sp. nov., a poly-ß-hydroxybutyrate-accumulating bacterium isolated from a freshwater pond.

A novel bacterium, designated strain Ruye-53(T), was isolated from a freshwater pond used to rear tilapiine cichlid fish in Taiwan. The taxonomic position of the novel strain was determined using a polyphasic approach. Strain Ruye-53(T) was Gram-reaction-negative, aerobic, cream-white coloured, rod-shaped, non-motile and poly-ß-hydroxybutyrate-accumulating. Growth occurred at 10-37 °C (optimum between 20 and 25 °C), at pH 6.0-9.0 (optimum between pH 8.0 and pH 9.0) and with 0-1 % (w/v) NaCl (optimum 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Ruye-53(T) belonged to the genus Gemmobacter and that its most closely related neighbour was Gemmobacter aquatilis DSM 3857(T) (97.6 % sequence similarity). The novel strain's predominant fatty acid was C18 : 1ω7c, its major respiratory quinone was Q-10 and its genomic DNA G+C content was 61.2 mol%. The polar lipid profile consisted of a mixture of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one uncharacterized aminolipid and four uncharacterized phospholipids. The DNA-DNA relatedness value between strain Ruye-53(T) and Gemmobacter aquatilis DSM 3857(T) was about 45.5 % (48.2±0.4 % in the reciprocal experiment). On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Ruye-53(T) represents a novel species in the genus Gemmobacter, for which the name Gemmobacter tilapiae sp. nov. is proposed. The type strain is Ruye-53(T) ( = BCRC 80261(T)  = KCTC 23310(T)).

Biosynthesis of branched-chain amino acids is essential for effective symbioses between betarhizobia and Mimosa pudica.

Burkholderia phymatum STM815 and Cupriavidus taiwanensis LMG19424 are betaproteobacterial strains that can effectively nodulate several species of the large legume genus Mimosa. A Tn5 mutant, derived from B. phymatum STM815 (KM60), and another derived from C. taiwanensis LMG19424 (KM184-55) induced Fix(-) nodules on Mimosa pudica. The Tn5-interrupted genes of the mutants showed strong homologies to ilvE, which encodes a branched-chain amino acid aminotransferase, and leuC, which encodes the large subunit of isopropylmalate isomerase. Both enzymes are known to be involved in the biosynthetic pathways for branched-chain amino acids (BCAAs) (leucine, valine and isoleucine). The B. phymatum ilvE mutant, KM60, was not auxotrophic for BCAAs and could grow well on minimal medium with pyruvate as a carbon source and ammonia as a nitrogen source. However, it grew less efficiently than the wild-type (WT) strain when ammonia was substituted with valine or isoleucine as a nitrogen source. The BCAA aminotransferase activity of KM60 was significantly reduced relative to the WT strain, especially with isoleucine and valine as amino group donors. The C. taiwanensis leuC mutant, KM184-55, could not grow on a minimal medium with pyruvate as a carbon source and ammonia as a nitrogen source, but its growth was restored when leucine was added to the medium. The isopropylmalate isomerase activity of KM184-55 was completely lost compared with the WT strain. Both mutants recovered their respective enzyme activities after complementation with the WT ilvE or leuC genes and were subsequently able to grow as well as their parental strains on minimal medium. They were also able to form nitrogen-fixing nodules on M. pudica. We conclude that the biosynthesis of BCAAs is essential for the free-living growth of betarhizobia, as well as for their ability to form effective symbioses with their host plant.

Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16ß), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(ß) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16ß) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16ß) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16ß) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

Effect of phosphoglycerate mutase and fructose 1,6-bisphosphatase deficiency on symbiotic Burkholderia phymatum.

Burkholderia phymatum STM815 is a ß-rhizobial strain that can effectively nodulate several species of the large legume genus Mimosa. Two Tn5-induced mutants of this strain, KM16-22 and KM51, failed to form root nodules on Mimosa pudica, but still caused root hair deformation, which is one of the early steps of rhizobial infection. Both mutants grew well in a complex medium. However, KM16-22 could not grow on minimal medium unless a sugar and a metabolic intermediate such as pyruvate were provided, and KM51 also could not grow on minimal medium unless a sugar was added. The Tn5-interrupted genes of the mutants showed strong homologies to pgm, which encodes 2,3-biphosphoglycerate-dependent phosphoglycerate mutase (dPGM), and fbp, which encodes fructose 1,6-bisphosphatase (FBPase). Both enzymes are known to be involved in obligate steps in carbohydrate metabolism. Enzyme assays confirmed that KM16-22 and KM51 had indeed lost dPGM and FBPase activity, respectively, whilst the activities of these enzymes were expressed normally in both free-living bacteria and symbiotic bacteroids of the parental strain STM815. Both mutants recovered their enzyme activity after the introduction of wild-type pgm or fbp genes, were subsequently able to use carbohydrate as a carbon source, and were able to form root nodules on M. pudica and to fix nitrogen as efficiently as the parental strain. We conclude that the enzymes dPGM and FBPase are essential for the formation of a symbiosis with the host plant.

Delftia litopenaei sp. nov., a poly-ß-hydroxybutyrate-accumulating bacterium isolated from a freshwater shrimp culture pond.

A Gram-negative, short-rod-shaped, motile, non-spore-forming and poly-ß-hydroxybutyrate-accumulating bacterial strain, designated wsw-7(T), was isolated from a freshwater shrimp culture pond in Taiwan and was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest relatives of strain wsw-7(T) were Delftia lacustris 332(T), Delftia tsuruhatensis T7(T) and Delftia acidovorans ATCC 15668(T), with sequence similarities of 98.5, 98.4 and 97.9%, respectively. Phylogenetic trees obtained with 16S rRNA gene sequences or the polyhydroxyalkanoate synthase (phaC) gene sequences revealed that strain wsw-7(T) and these three closest relatives formed an independent phylogenetic clade within the order Burkholderiales. Strain wsw-7(T) contained summed feature 3 (comprising C(16:1)ω7c and/or C(16:1)ω6c), C(18:0) and C(18:1)ω7c as predominant fatty acids. The major isoprenoid quinone was Q-8 and the DNA G+C content was 67.6 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an uncharacterized aminolipid and several uncharacterized phospholipids. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain wsw-7(T) represents a novel species in the genus Delftia, for which the name Delftia litopenaei sp. nov. is proposed; the type strain is wsw-7(T) ( = BCRC 80212(T) = LMG 25724(T)).