Rapid and Selective Screening Method for Isolation and Identification of Carotenoid-Producing Bacteria.

Carotenoids are naturally occurring yellow to red pigments with many biological activities including antioxidant, anticancer, anti-inflammatory, membrane stabilizers, and precursors for vitamin A. These biological activities are linked with many health benefits (e.g., anticarcinogenic activity, prevention of chronic diseases, etc.), which grew the interest of several industrial sectors especially in food, feed, nutraceuticals, cosmetics, and pharmaceutical industries. The production of natural carotenoids from microbial sources such as bacteria can help meet the growing global market of carotenoids estimated at $1.5 billion in 2014 and is expected to reach 1.8 billion in 2019. This chapter demonstrates, step-by-step, the development of a rapid and selective screening method for isolation and identification of carotenoid-producing microorganisms and their carotenoid analysis. This method involves three main procedures: UV treatment, sequencing analysis of 16S rRNA genes, and carotenoids analysis using rapid and effective HPLC-diode array-MS methods.

Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens.

The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17T) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140-148, 2007).In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17T. We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.

LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

A novel radio-tolerant astaxanthin-producing bacterium reveals a new astaxanthin derivative: astaxanthin dirhamnoside.

Astaxanthin is a red ketocarotenoid that exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster. The recent discovery of the beneficial roles of astaxanthin against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue has raised its market demand as a nutraceutical and medicinal ingredient in aquaculture, food, and pharmaceutical industries. To satisfy the growing demand for this high-value nutraceuticals ingredient and consumer interest in natural products, many research efforts are being made to discover novel microbial producers with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diodearray-MS methods for carotenoids analysis, we succeeded to isolate a unique astaxanthin-producing bacterium (strain TDMA-17(T)) that belongs to the family Sphingomonadaceae (Asker et al., Appl Microbiol Biotechnol 77: 383-392, 2007). In this chapter, we provide a detailed description of effective HPLC-Diodearray-MS methods for rapid analysis and identification of the carotenoids produced by strain TDMA-17(T). We also describe the methods of isolation and identification for a novel bacterial carotenoid (astaxanthin derivative), a major carotenoid that is produced by strain TDMA-17(T). Finally, we describe the polyphasic taxonomic analysis of strain TDMA-17(T) and the description of a novel species belonging to genus Sphingomonas.

Isolation, characterization, and diversity of novel radiotolerant carotenoid-producing bacteria.

Carotenoids are natural pigments that exhibit many biological functions, such as antioxidants (i.e., promote oxidative stress resistance), membrane stabilizers, and precursors for vitamin A. The link between these biological activities and many health benefits (e.g., anticarcinogenic activity, prevention of chronic diseases, etc.) has raised the interest of several industrial sectors, especially in the cosmetics and pharmaceutical industries. The use of microorganisms in biotechnology to produce carotenoids is favorable by consumer and can help meet the growing demand for these bioactive compounds in the food, feed, and pharmaceutical industries. This methodological chapter details the development of a rapid and selective screening method for isolation and identification of carotenoid-producing microorganisms based on UV treatment, sequencing analysis of 16S rRNA genes, and carotenoids' analysis using rapid and effective High-Performance Liquid Chromatography-Diodearray-MS methods. The results of a comprehensive 16S rRNA gene-based phylogenetic analysis revealed a diversity of carotenoid-producing microorganisms (104 isolates) that were isolated at a high frequency from water samples collected at Misasa (Tottori, Japan), a region known for its high natural radioactivity content. These carotenoid-producing isolates were classified into 38 different species belonging to 7 bacterial classes (Flavobacteria, Sphingobacteria, α-Proteobacteria, γ-Proteobacteria, Deinococci, Actinobacteria, and Bacilli). The carotenoids produced by the isolates were zeaxanthin (6 strains), dihydroxyastaxanthin (24 strains), astaxanthin (27 strains), canthaxanthin (10 strains), and unidentified molecular species that were produced by the isolates related to Deinococcus, Exiguobacterium, and Flectobacillus. Here, we describe the methods used to isolate and classify these microorganisms.

Novel zeaxanthin-producing bacteria isolated from a radioactive hot spring water.

Zeaxanthin is a powerful antioxidant that is widely found in vegetables and fruits. Epidemiological evidences suggest that increasing the consumption of zeaxanthin in the diet is associated with a lower risk of age-related macular degeneration, helps prevent glaucoma and cataracts, and supports normal eye health. Zeaxanthin is a promising nutraceutical with many applications in the feed, food, and pharmaceutical industries. Currently, the commercial production of zeaxanthin is still dependant on synthetic routes with limitation for the biological one. Nevertheless, the biotechnological production of zeaxanthin is emerging due to its safety, potential large-scale production, and consumers' demand and preference for natural additives. Using a rapid screening method based on 16S rRNA gene and effective high-performance liquid chromatography (HPLC)-Diodearray-MS methods for carotenoids' analysis, we isolated effective zeaxanthin-producing bacteria (strain TDMA-5(T) and -16(T)) that belong to the family Sphingobacteriaceae and Sphingomonadaceae, respectively. In this chapter, we provide a detailed description of the HPLC-Diodearray-MS methods used for rapid analysis and identification of the carotenoids produced by both strains. In addition, the polyphasic taxonomic analysis of both novel strains and the description of a novel species and genus are described.

Deinococcus depolymerans sp. nov., a gamma- and UV-radiation-resistant bacterium, isolated from a naturally radioactive site.

Four gamma- and UV-radiation-resistant bacterial strains, designated TDMA-24(T), TDMA-24-2, TDMA-24-3 and TDMA-24-4, were isolated from a fresh-water sample collected at Misasa, Tottori, Japan. Cells of these strains were Gram-reaction-positive, non-motile, non-spore-forming, rod-shaped and formed red colonies. The genomic DNA G+C contents ranged from 70.5 to 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolates belong to the genus Deinococcus, the highest sequence similarities being with Deinococcus aquaticus PB314(T) (98%) and Deinococcus caeni Ho-08(T) (97 %). The polar lipid profile of strain TDMA-24(T) comprised three unidentified phosphoglycolipids, five unidentified glycolipids and seven unidentified polar lipids. MK-8 was the predominant respiratory quinone. Major fatty acids were iso-C(15 : 0), C(15 : 1)ω6c, C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c). On the basis of their phylogenetic positions and chemotaxonomic and phenotypic characteristics, the novel isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus depolymerans sp. nov. is proposed. The type strain is TDMA-24(T) ( = JCM 14369(T)  = NBRC 102115(T)  = CCUG 53609(T)).

Deinococcus aquiradiocola sp. nov., isolated from a radioactive site in Japan.

A gamma- and UV-radiation-tolerant, pale-pink strain (TDMA-uv53T) was isolated from a freshwater sample collected at Misasa (Tottori, Japan), after exposure of the water sample to UV radiation. The cells stained Gram-positive and were non-motile, rod-shaped and non-spore-forming. The DNA G+C content of the strain was 69.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TDMA-uv53T belongs to the genus Deinococcus, the highest sequence similarities being found with Deinococcus claudionis PO-04-19-125T (96 %), D. altitudinis ME-04-01-32T (96 %), D. radiomollis PO-04-20-132T (95 %), D. deserti VCD115T (91.5 %), D. hopiensis KR-140T (91.0 %) and D. sonorensis KR-87T (91.0 %). Major fatty acids were iso-15 : 0, 15 : 1omega6c, 15 : 0, 16 : 0 and summed feature 3 (iso-15 : 0 2-OH and/or 16 : 1omega7c). MK-8 was the predominant respiratory quinone. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-uv53T from closely related Deinococcus species. The results of our polyphasic taxonomic analyses suggested that TDMA-uv53T represents a novel Deinococcus species, for which the name Deinococcus aquiradiocola sp. nov. is proposed. The type strain is TDMA-uv53T (=JCM 14371(T) [corrected] =NBRC 102118T =CCUG 53612T).

Lutispora thermophila gen. nov., sp. nov., a thermophilic, spore-forming bacterium isolated from a thermophilic methanogenic bioreactor digesting municipal solid wastes.

A novel anaerobic, moderately thermophilic, spore-forming, rod-shaped bacterium (strain EBR46T) was isolated from an enrichment culture derived from an anaerobic thermophilic (55 degrees C) methanogenic bioreactor treating artificial solid wastes. Phylogeny based on 16S rRNA gene sequence analysis placed strain EBR46T within a distinct lineage between Clostridium clusters II and III. The closest recognized relative of strain EBR46T was Gracilibacter thermotolerans DSM 17427T (85.3 % 16S rRNA gene sequence similarity). The DNA G+C content of strain EBR46T was 36.2 mol%. The novel strain grew optimally at 55-58 degrees C and at pH 7.5-8.0 and was able to grow on peptone, tryptone, Casamino acids, casein hydrolysate, methionine, threonine, tryptophan, cysteine, lysine and serine in the presence of 0.2 % yeast extract. Carbohydrates were not utilized. The main products from tryptone utilization were acetate, iso-butyrate, propionate and iso-valerate. Strain EBR46T produced hydrogen sulfide from cysteine. The major fatty acids were iso-C15 : 0, C14 : 0, C16 : 0 DMA (dimethyl acetal) and iso-C15 : 0 DMA. Based on its unique phylogenetic and physiological features, strain EBR46T is considered to represent a novel species of a new genus, for which the name Lutispora thermophila gen. nov., sp. nov. is proposed. The type strain of the type species is EBR46T (=NBRC 102133T=DSM 19022T).

Sphingomonas jaspsi sp. nov., a novel carotenoid-producing bacterium isolated from Misasa, Tottori, Japan.

A yellow-pigmented, Gram-negative, motile, strictly aerobic, pleomorphic bacterium (strain TDMA-16(T)) was isolated from a freshwater sample collected at Misasa (Tottori, Japan). Strain TDMA-16(T) was slightly tolerant to gamma-ray irradiation and produced carotenoids, including zeaxanthin, nostoxanthin and an unknown carotenoid, effectively [1.7 mg (g dry cells)(-1)]. The DNA G+C content of strain TDMA-16(T) was 63.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed strain TDMA-16(T) in a distinct lineage in the family Sphingomonadaceae; sequence data showed that strain TDMA-16(T) was most closely related to Sphingomonas mali IFO 15500(T) (95.1 %), Sphingomonas aquatilis JSS7(T) (95.0 %), Sphingomonas pruni IFO 15498(T) (94.9 %), Sphingomonas melonis DSM 14444(T) (94.9 %) and Sphingomonas asaccharolytica IFO 15499(T) (94.5 %). The major fatty acids of strain TDMA-16(T) were C(17 : 1) omega 6c (34.5 %) and C(18 : 1) omega 7c (29.3 %). The presence of Q-10 as the main ubiquinone, the Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profile and 2-hydroxy fatty acids, plus the absence of 3-hydroxy fatty acids, supported identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-16(T) from closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggest that strain TDMA-16(T) represents a novel Sphingomonas species, for which the name Sphingomonas jaspsi sp. nov. is proposed. The type strain is strain TDMA-16(T) (=NBRC 102120(T)=DSM 18422(T)=CCUG 53607(T)).

Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan.

A red-pigmented, Gram-negative, motile, strictly aerobic, mesophilic, oval- or short rod-shaped bacterium (TDMA-17(T)) was isolated from fresh water collected at Misasa, a radioactive site in Japan. TDMA-17(T) was slightly tolerant against gamma-ray irradiation, and effectively produced carotenoids (2.8 mg g(-1) dry cells) including, astaxanthin and astaxanthin isomers. Phylogenetic analysis based on 16S rRNA gene sequences placed TDMA-17(T) in a distinct lineage in the family Sphingomonadaceae, and the highest degree of sequence similarity determined were to Sphingomonas aerolata NW12(T) (94.5%), Sphingomonas aurantiaca MA101b(T) (94.0%), Sphingomonas melonis DAPP-PG 224(T) (94.0%), Sphingomonas asaccharolytica IFO 15499(T) (93.9%) and Sphingomonas abaci C42(T) (93.9%). The major fatty acids were C(17 : 1)omega6c (33.0%) and C(18 : 1)omega7c (20.8%). The DNA G+C content was 67.7 mol%. The presence of Q-10 as the main ubiquinone, the presence of Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profiles, the presence of 2-hydroxy fatty acids and the absence of 3-hydroxy fatty acids supported the identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-17(T) from the closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggested that TDMA-17(T) represents a novel Sphingomonas species, for which the name Sphingomonas astaxanthinifaciens sp. nov. is proposed. The type strain is TDMA-17(T) (=NBRC 102146=CCUG 53608).

Mesoflavibacter zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthin-producing marine bacterium of the family Flavobacteriaceae.

A novel strictly aerobic, gliding, Gram-negative, rod-shaped, halo- and mesophilic bacterium (TD-ZX30(T)) was isolated from a seawater sample collected on the Pacific coastline of Japan near Kamakura City (Fujisawa, Kanagawa). The temperature range for growth of TD-ZX30(T) was between 16 and 44 degrees C. The DNA G+C content was 32.0mol%. The predominant fatty acids were iso-C(15:1) G, iso-C(15:0), iso-C(16:0) 3-OH, iso-C(15:0) 3-OH, Summed feature (iso-C(15:0) 2-OH and/or C(16:1)omega7c), iso-C(17:0) 3-OH, and C(15:0). MK-6 was the only respiratory quinone. Zeaxanthin was the major carotenoid pigment produced but flexirubin-type pigments were not produced. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that TD-ZX30(T) belonged to a distinct lineage in the family Flavobacteriaceae, sharing 93.9% sequence similarity with the nearest species Olleya marilimosa. TD-ZX30(T) could be distinguished from the other members of the family Flavobacteriaceae by a number of chemotaxonomic and phenotypic characteristics. The results of polyphasic taxonomic analyses suggested that TD-ZX30(T) represents a novel genus and a novel species, for which the name Mesoflavibacter zeaxanthinifaciens gen. nov., sp. nov. is proposed. The type strain is TD-ZX30(T) (=NBRC 102119=CCUG 53614=DSM 18436).

Proteins encoded by the conservon of Streptomyces coelicolor A3(2) comprise a membrane-associated heterocomplex that resembles eukaryotic G protein-coupled regulatory system.

Streptomyces coelicolor A3(2) retains unique conserved operons termed conservons. Here, one of the conservons (cvn9), which encodes five proteins (A9-E9), was characterized. Mutants for cvnA9 and cvnAlO conditionally overproduced actinorhodin and performed precocious aerial growth, while a cvnE9 mutant showed the parental phenotype. Transcription of bidG, adpA and bldN was upregulated in the cvnA9 mutant. A9-D9 were detected in the insoluble fraction of cell-free extract of S. coelicolor by Western analysis. Biochemical analyses revealed that A9 has ATP-hydrolysing and adenine nucleotide-binding activities; D9 has GTP-hydrolysing and guanine nucleotide-binding activities; and E9 shows a typical spectrum similar to cytochrome P450. The comprehensive interaction assays demonstrated the occurrence of specific interactions between A9 and B9, A9 and C9, B9 and B9, B9 and D9, and C9 and D9. A9 associated with and dissociated from B9 (and C9) when ATP and ATP-gamma-S were supplied in the reaction respectively. Similarly, D9 associated with and dissociated from B9 (and C9) when GTP and GTP-gamma-S were supplied respectively. A9 and B9 were also shown for the occurrence as homocomplexes. Probably, Cvn9 proteins comprise a membrane-associated heterocomplex resembling the eukaryotic G-protein-coupled receptor system, which may serve as a signal transducer that connects to the bld cascade.

AmfS, an extracellular peptidic morphogen in Streptomyces griseus.

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.