High-level production of poly-γ-glutamic acid from untreated molasses by Bacillus siamensis IR10.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer and has been applied in many fields. Bacillus siamensis SB1001 was a newly isolated poly-γ-glutamic acid producer with sucrose as its optimal carbon source. To improve the utilization of carbon source, and then molasses can be effectively used for γ-PGA production, 60cobalt gamma rays was used to mutate the genes of B. siamensis SB1001. RESULTS: Bacillus siamensis IR10 was screened for the production of γ-PGA from untreated molasses. In batch fermentation, 17.86 ± 0.97 g/L γ-PGA was obtained after 15 h, which is 52.51% higher than that of its parent strain. Fed-batch fermentation was performed to further improve the yield of γ-PGA with untreated molasses, yielding 41.40 ± 2.01 g/L of γ-PGA with a productivity of 1.73 ± 0.08 g/L/h. An average γ-PGA productivity of 1.85 g/L/h was achieved in the repeated fed-batch fermentation. This is the first report of such a high γ-PGA productivity. The analysis of the enzyme activities showed that they were affected by the carbon sources, enhanced ICDH and GDH, and decreased ODHC, which are important for γ-PGA production. CONCLUSION: These results suggest that untreated molasses can be used for economical and industrial-scale production of γ-PGA by B. siamensis IR10.

Simultaneous production of poly-γ-glutamic acid and 2,3-butanediol by a newly isolated Bacillus subtilis CS13.

Bacillus subtilis naturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-γ-glutamic acid (γ-PGA) production. 2,3-BD is a promising platform chemical in various industries, and co-production of the two chemicals has great economic benefits. Co-production of γ-PGA and 2,3-BD by a newly isolated B. subtilis CS13 was investigated here. The fermentation medium and culture parameters of the process were optimized using statistical methods. It was observed that sucrose, L-glutamic acid, ammonium citrate, and MgSO4·7H2O were favorable for γ-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 °C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/L L-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO4·7H2O was obtained by response surface methodology (RSM). The results show that the titers of γ-PGA and 2,3-BD reached 27.8 ± 0.9 g/L at 24 h and 57.1 ± 1.3 g/L at 84 h with the optimized medium, respectively. γ-PGA and 2,3-BD production by B. subtilis CS13 was significantly enhanced in fed-batch fermentations. γ-PGA (36.5 ± 1.1 g/L, productivity of 1.22 ± 0.04 g/L/h) and 2,3-BD concentrations (119.6 ± 2.8 g/L, productivity of 2.49 ± 0.66 g/L/h) were obtained in the optimized medium with feeding sucrose. The co-production of 2,3-BD and γ-PGA provides a new perspective for industrial production of γ-PGA and 2,3-BD. Key points • A strategy for co-production of γ-PGA and 2,3-BD was developed. • The culture parameters for the co-production of γ-PGA and 2,3-BD were studied. • RSM was used to optimize the medium for γ-PGA and 2,3-BD co-production. • 36.5 g/L γ-PGA and 119.6 g/L 2,3-BD were obtained from the optimum medium in fed-batch fermentation.

Deinococcus radioresistens sp. nov., a UV and gamma radiation-resistant bacterium isolated from mountain soil.

Two Gram-negative, non-motile, short rod-shaped bacterial strains, designated as 8A(T) and 28A, were isolated from Mount Deogyusan, Jeonbuk Province, South Korea. The isolates were analyzed by a polyphasic approach, revealing variations in their phenotypic characters but high DNA-DNA hybridisation values reciprocally, confirming that they belong to the same species. Both the isolates also showed a high resistance to UV compared with Deinococcus radiodurans, and a gamma-radiation resistance similar to other members of the genus Deinococcus. Phylogenetic analysis with the 16S rRNA gene sequences of closely related species indicated their similarities were below 97 %. Chemotaxonomic data showed the most abundant fatty acids to be C16:1ω7c and C16:0. The strains can be distinguished from closely related species by the production of esterase (C4) and α-galactosidase, and by their ability to assimilate L-alanine, L-histidine and N-acetyl-D-glucosamine. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus radioresistens sp. nov. is proposed. The type strain is 8A(T) (KEMB 9004-109(T) = JCM 19777(T)), and a second strain is 28A (KEMB 9004-113 = JCM 19778).

Deinococcus puniceus sp. nov., a bacterium isolated from soil-irradiated gamma radiation.

A Gram-positive, coccus-shaped, crimson-color-pigmented bacterium was isolated from soil irradiated with 5 kGy gamma radiation and was designated strain DY1(T). Cells showed growth at 10-30 °C and pH 7-11 and were oxidase-negative and catalase-positive. Phylogenetic analyses of the 16S rRNA gene showed that the strain DY1(T) belonged to the genus Deinococcus with sequence similarities to Deinococcus aquatilis CCUG 53370(T) (96.2 %) and Deinococcus navajonensis KR-114(T) (94.1 %). Strain DY1(T) showed low level of DNA relatedness with D. aquatilis CCUG 53370(T) (41.3 ± 3.9 %). The DNA G + C content of DY1(T) was 58.7 mol%. Predominant fatty acids were summed feature 3 (C16:1 ω7c/ω6c), C16:0, and C17:0. The major amino acids were D-alanine, L-glutamic acid, glycine, and L-ornithine in the peptidoglycan. The major polar lipids were unknown phosphoglycolipids (PGL). Strain DY1(T) has resistance to gamma radiation and was found to be a novel species. Therefore, the strain was designated as DY1(T) (=KCTC 33027(T) = JCM 18576(T)), and the name Deinococcus puniceus sp. nov. is herein proposed.

Spirosoma radiotolerans sp. nov., a gamma-radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A(T), was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A(T) belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74(T), 93.4 % with Spirosoma rigui WPCB118(T), 92.8 % with Spirosoma luteum SPM-10(T), 92.7 % with Spirosoma spitsbergense SPM-9(T), and 91.9 % with Spirosoma panaciterrae Gsoil 1519(T). Strain DG5A(T) revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C(16:1) ω7c/C(16:1) ω6c (36.90 %), C(16:1) ω5c (29.55 %), and iso-C(15:0) (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A(T) was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A(T) presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A(T) (=KCTC 32455(T) = JCM19447(T)).

Hymenobacter swuensis sp. nov., a gamma-radiation-resistant bacteria isolated from mountain soil.

Gram stain-negative and non-motile bacteria, designated as DY53(T) and DY43, were isolated from mountain soil in South Korea prior exposure with 5 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strains belonged to the family Cytophagaceae in the class Cytophagia. 16S rRNA gene sequence similarity of strains DY53(T) and DY43 was 100 %. The highest degrees of sequence similarities of strains DY53(T) and DY43 were found with Hymenobacter perfusus A1-12(T) (98.8 %), Hymenobacter rigui WPCB131(T) (98.5 %), H. yonginensis HMD1010(T) (97.9 %), H. xinjiangensis X2-1g(T) (96.6 %), and H. gelipurpurascens Txg1(T) (96.5 %). The DNA G+C content of the novel strains DY53(T) and DY43 were 59.5 mol%. Chemotaxonomic data revealed that strains possessed major fatty acids such as C15:0 iso, C15:0 anteiso, C16:1 ω5c, summed feature 3 (16:1 ω7c/ω6c), summed feature 4 (17:1 anteiso B/iso I) and C17:0 iso, and major polar lipid was phosphatidylethanolamine. The novel strains showed resistance to gamma radiation, with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) in excess of 5 kGy. Based on these data, strains DY53(T) and DY43 should be classified as representing a novel species, for which the name Hymenobacter swuensis sp. nov. is proposed, with the type strain DY53(T) (=KCTC 32018(T) = JCM 18582(T)) and DY43 (=KCTC 32010).

Enhancement of 1,3-propanediol production by expression of pyruvate decarboxylase and aldehyde dehydrogenase from Zymomonas mobilis in the acetolactate-synthase-deficient mutant of Klebsiella pneumoniae.

The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol.

Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding.

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.

FrnE, a cadmium-inducible protein in Deinococcus radiodurans, is characterized as a disulfide isomerase chaperone in vitro and for its role in oxidative stress tolerance in vivo.

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ~15- and ~3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ~2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42 °C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation.

The role of aldehyde/alcohol dehydrogenase (AdhE) in ethanol production from glycerol by Klebsiella pneumoniae.

Transcriptome analysis of a K. pneumoniae GEM167 mutant strain derived by irradiation with gamma rays, which exhibited high-level production of ethanol from glycerol, showed that the mutant expressed AdhE at a high level. Ethanol production decreased significantly, from 8.8 to 0.5 g l(-1), when an adhE-deficient derivative of that strain was grown on glycerol. Bacterial growth was also reduced under such conditions, showing that AdhE plays a critical role in maintenance of redox balance by catalyzing ethanol production. Overexpression of AdhE enhanced ethanol production, from pure or crude glycerol, to a maximal level of 31.9 g l(-1) under fed-batch fermentation conditions; this is the highest level of ethanol production from glycerol reported to date.

Comparative survival analysis of 12 histidine kinase mutants of Deinococcus radiodurans after exposure to DNA-damaging agents.

Bacteria are able to adapt to changes in the environment using two-component signal transduction systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). Deinococcus radiodurans, one of the most resistant organisms to ionizing radiation, has 20 putative HKs and 25 putative RRs. In this study, we constructed 12 D. radiodurans mutant strains lacking a gene encoding a HK and surveyed their resistance to γ-radiation, UV-B radiation (302 nm), mitomycin C (MMC), and H(2)O(2). Five (dr0860 (-), dr1174 (-), dr1556 (-), dr2244 (-), and dr2419 (-)) of the 12 mutant strains showed at least a one-log cycle reduction in γ-radiation resistance. The mutations (1) dr1174, dr1227, and dr2244 and (2) dr0860, dr2416, and dr2419 caused decreases in resistance to UV radiation and MMC, respectively. Only the dr2416 and dr2419 mutant strains showed higher sensitivity to H(2)O(2) than the wild-type. Reductions in the resistance to γ-radiation and H(2)O(2), but not to UV and MMC, were observed in the absence of DR2415, which seems to be a cognate RR of DR2416. This result suggests that DR2415/DR2416 (DrtR/S: DNA damage response TCS) may be another TCS responsible for the extreme resistance of D. radiodurans to DNA-damaging agents.

Deinococcus daejeonensis sp. nov., isolated from sludge in a sewage disposal plant.

A Gram-stain-positive, strictly aerobic, spherical, non-motile red-pigmented bacterial strain, designated MJ27(T), was isolated from a sludge sample of the Daejeon sewage disposal plant in South Korea. A polyphasic approach was used to study the taxonomic position of strain MJ27(T). Strain MJ27(T) shared highest 16S rRNA gene sequence similarity with Deinococcus grandis DSM 3963(T) (98.8 %), Deinococcus caeni Ho-08(T) (97.5 %) and Deinococcus aquaticus PB314(T) (96.6 %.); levels of sequence similarity with the type strains of other Deinococcus species were less than 96.0 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ27(T) belonged to the clade formed by members of the genus Deinococcus in the family Deinococcaceae. The G+C content of the genomic DNA of strain MJ27(T) was 67.6 mol%. The chemotaxonomic characteristics of strain MJ27(T) were typical of members of the genus Deinococcus, with MK-8 as the predominant respiratory quinone, C(16:1)ω7c, C(15:1)ω6c, C(16:0) and C(15:0) as major fatty acids (>12 %), ornithine as the diamino acid in the cell-wall peptidoglycan and resistance to gamma radiation [D(10) (dose required to reduce the bacterial population by tenfold) >9 kGy]. The low levels of DNA-DNA relatedness reported here (5.3±1.5-29.2±2.3 %) indicate that strain MJ27(T) represents a species that is separate from its closest relatives in the genus Deinococcus. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ27(T) is considered to represent a novel species of the genus Deinococcus, for which the name Deinococcus daejeonensis sp. nov. is proposed. The type strain is MJ27(T) ( = KCTC 13751(T) = JCM 16918(T)).

Deinococcus humi sp. nov., isolated from soil.

A Gram-staining-positive, strictly aerobic, spherical, non-motile, red-pigmented bacterium, designated strain MK03(T), was isolated from a soil sample collected in South Korea. The taxonomic position of the novel strain was investigated using a polyphasic approach. In phylogenetic analyses based on 16S rRNA gene sequences, strain MK03(T) was placed in a clade formed by members of the genus Deinococcus in the family Deinococcaceae and appeared to be most closely related to Deinococcus aerolatus 5516T-9(T) (97.4% sequence similarity), Deinococcus marmoris AA-63(T) (97.2%), Deinococcus radiopugnans ATCC 19172(T) (97.2%) and Deinococcus saxicola AA-1444(T) (96.9%). The genomic DNA G+C content of the novel strain was 64.5 mol%. The chemotaxonomic characteristics of strain MK03(T) were typical of members of the genus Deinococcus: MK-8 was identified as the predominant respiratory quinine, the major fatty acids were C(16:1)ω7c, C(15:1)ω6c, C(16:0) and C(15:0, )ornithine was found to be the diamino acid in the cell-wall peptidoglycan and the novel strain showed resistance to gamma radiation, with a D(10) value (i.e. the dose required to reduce the bacterial population by 10-fold) in excess of 9 kGy. In hybridization experiments, only low DNA-DNA relatedness values (11.6-34.5%) were recorded between the novel strain and its closest relatives in the genus Deinococcus. Based on the phylogenetic, chemotaxonomic, phenotypic and DNA-DNA relatedness data, strain MK03(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus humi sp. nov. is proposed. The type strain is MK03(T) ( = KCTC 13619(T)  = JCM 17915(T)).

Pigment-based whole-cell biosensor system for cadmium detection using genetically engineered Deinococcus radiodurans.

In this study, a colorimetric whole-cell biosensor for cadmium (Cd) was designed using a genetically engineered red pigment producing bacterium, Deinococcus radiodurans. Based on the previous microarray data, putative promoter regions of highly Cd-inducible genes (DR_0070, DR_0659, DR_0745, and DR_2626) were screened and used for construction of lacZ reporter gene cassettes. The resultant reporter cassettes were introduced into D. radiodurans R1 to evaluate promoter activity and specificity. Among the promoters, the one derived from DR_0659 showed the highest specificity, sensitivity, and activity in response to Cd. The Cd-inducible activity was retained in the 393-bp deletion fragment (P0659-1) of the P0569 promoter, but the expression pattern of the putative promoter fragments inferred its complex regulation. The detection range was from 10 to 1 mM of Cd. The LacZ expression was increased up to 100 µM of Cd, but sharply decreased at higher concentrations. For macroscopic detection, the sensor plasmid (pRADI-P0659-1) containing crtI as a reporter gene under the control of P0659-1 was introduced into a crtI-deleted mutant strain of D. radiodurans (KDH018). The color of this sensor strain (KDH081) changed from light yellow to red by the addition of Cd and had no significant response to other metals. Color change by the red pigment synthesis could be clearly recognized in a day with the naked eye and the detection range was from 50 nM to 1 mM of Cd. These results indicate that genetically engineered D. radiodurans (KDH081) can be used to monitor the presence of Cd macroscopically.

Process optimization for mass production of 2,3-butanediol by Bacillus subtilis CS13.

BACKGROUND: Bacillus subtilis CS13 was previously isolated for 2,3-butanediol (2,3-BD) and poly-γ-glutamic acid (γ-PGA) co-production. When culturing this strain without L-glutamic acid in the medium, 2,3-BD is the main metabolic product. 2,3-BD is an important substance and fuel with applications in the chemical, food, and pharmaceutical industries. However, the yield and productivity for the B. subtilis strain should be improved for more efficient production of 2,3-BD. RESULTS: The medium composition, which contained 281.1 g/L sucrose, 21.9 g/L ammonium citrate, and 3.6 g/L MgSO4·7H2O, was optimized by response surface methodology for 2,3-BD production using B. subtilis CS13. The maximum amount of 2,3-BD (125.5 ± 3.1 g/L) was obtained from the optimized medium after 96 h. The highest concentration and productivity of 2,3-BD were achieved simultaneously at an agitation speed of 500 rpm and aeration rate of 2 L/min in the batch cultures. A total of 132.4 ± 4.4 g/L 2,3-BD was obtained with a productivity of 2.45 ± 0.08 g/L/h and yield of 0.45 g2,3-BD/gsucrose by fed-batch fermentation. The meso-2,3-BD/2,3-BD ratio of the 2,3-BD produced by B. subtilis CS13 was 92.1%. Furthermore, 89.6 ± 2.8 g/L 2,3-BD with a productivity of 2.13 ± 0.07 g/L/h and yield of 0.42 g2,3-BD/gsugar was achieved using molasses as a carbon source. CONCLUSIONS: The production of 2,3-BD by B. subtilis CS13 showed a higher concentration, productivity, and yield compared to the reported generally recognized as safe 2,3-BD producers. These results suggest that B. subtilis CS13 is a promising strain for industrial-scale production of 2,3-BD.

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation.

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.

Complete genome sequence of Planococcus sp. PAMC21323 isolated from Antarctica and its metabolic potential to detoxify pollutants.

The Planococcus sp. PAMC21323 is a yellow pigment-producing bacterium isolated from King George Island in Antarctica; it has a broad growth temperature range of 5-40 °C. Herein, we describe the complete genome sequence information of the genus Planococcus with its annotated sequence, genetic features for bioremediation, and oxidative stress capacity. The Planococcus sp. PAMC21323 possesses chromosomal DNA (3,196,500-bp) with plasmid DNA (3364-bp). The complete 3,199,864-bp of the genome consists of 3171 genes including 60 transfer RNAs and 24 ribosomal RNAs. Strain PAMC21323 encodes various genes associated with detoxification of heavy metal ions and aromatic hydrocarbons. Moreover, it is equipped with diverse stress response systems, which can be used to sense the internal and oxidative stresses caused by detoxification. This is the first report highlighting the genetic potential of Planococcus sp. PAMC21323 in bioremediation, suggesting application of this psychrotrophic strain in bioremediation in harsh environments.

Isolation of a novel plasmid, pNi15, from Enterobacter sp. Ni15 containing a nickel resistance gene.

We isolated nickel-resistant bacterium from soil in order to identify a novel nickel resistance determinant. Using 16S rRNA gene sequencing, an isolate was identified as Enterobacter sp. Ni15. This species showed a medium-level (resistant to up to 10 mM) nickel resistance in nutrient-rich media. Enterobacter sp. Ni15 has a novel plasmid, pNi15, and an increased nickel resistance to Escherichia coli DH5alphain trans. To isolate the nickel resistance gene from pNi15, the plasmid was digested with XbaI and its fragments were cloned into pBluescriptIISK(+). The clones were transferred into E. coli DH5alpha. The nickel resistance of the clones was then assayed. From these results, a pNi15100 isolate containing a 5,328 bp XbaI fragment of pNi15 was identified and sequenced. The E. coli DH5alpha harboring the pNi15100 showed a resistance to up to 7 mM nickel. Using a subcloning analysis, we were able to identify the novel nickel resistance determinant: the nrp gene encoding the putative proteins NrpA and NrpB.

Microalgal lipid production using the hydrolysates of rice straw pretreated with gamma irradiation and alkali solution.

BACKGROUND: Lignocellulosic biomass has long been recognized as a potential sustainable source of sugars for biofuels. However, many physicochemical structural and compositional factors inhibit the enzymatic digestibility of the lignocellulosic biomass. In this study, efficient pretreatment method of rice straw (RS) was developed and the RS hydrolysate was applied in the cultivation of microalgae for lipid production. RESULTS: Gamma ray irradiation (GRI) and alkali solution were used for the pretreatment, and saccharification was carried out with lignocellulolytic enzymes. When RS was pretreated by combined GRI and alkali method, the glucose and xylose saccharification yield after enzymatic hydrolysis increased up to 91.65 and 98.84 %, respectively. The enzymatic hydrolysate from the RS pretreated with the combined method was used to cultivate Chlorella protothecoides for lipid production. The maximum concentrations of biomass and fatty acid methyl ester of cells were 6.51 and 2.95 g/L, respectively. The lipid content of C. protothecoides from RS hydrolysate was comparable to that from glucose, and the lipid composition was similar between different carbon sources. CONCLUSION: These results demonstrate that the combined pretreatment with gamma irradiation was highly effective in preparing hydrolysate, and the rice straw hydrolysate could be used as an alternative carbon source for microalgal lipid production for biofuel.

Expression and mutational analysis of DinB-like protein DR0053 in Deinococcus radiodurans.

In order to understand the mechanism governing radiation resistance in Deinococcus radiodurans, current efforts are aimed at identifying potential candidates from a large repertoire of unique Deinococcal genes and protein families. DR0053 belongs to the DinB/YfiT protein family, which is an over-represented protein family in D. radiodurans. We observed that dr0053 transcript levels were highly induced in response to gamma radiation (γ-radiation) and mitomycin C (MMC) exposure depending on PprI, RecA and the DrtR/S two-component signal transduction system. Protein profiles demonstrated that DR0053 is a highly induced protein in cultures exposed to 10 kGy γ-radiation. We were able to determine the transcriptional start site of dr0053, which was induced upon irradiation, and to assign the 133-bp promoter region of dr0053 as essential for radiation responsiveness through primer extension and promoter deletion analyses. A dr0053 mutant strain displayed sensitivity to γ-radiation and MMC exposure, but not hydrogen peroxide, suggesting that DR0053 helps cells recover from DNA damage. Bioinformatic analyses revealed that DR0053 is similar to the Bacillus subtilis protein YjoA, which is a substrate of bacterial protein-tyrosine kinases. Taken together, the DNA damage-inducible (din) gene dr0053 may be regulated at the transcriptional and post-translational levels.