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.

Screening, Isolation, and Identification of Zeaxanthin-Producing Bacteria.

Zeaxanthin is a yellow xanthophyll, dihydroxy-carotenoid, that is naturally found in some of the green, orange, and yellow vegetables and fruits and has a powerful antioxidant activity. Epidemiological evidences suggest that increasing the consumption of zeaxanthin in the diet is associated with a lower risk of age-related macular degeneration (ARMD) and cataracts, two of the leading causes of blindness in the world. Zeaxanthin is a promising nutraceutical/colorant with many applications in feed, food, and pharmaceutical industries. Currently, the commercial production of zeaxanthin is dependent on synthetic routes with limitation in production from biological sources. However, the biotechnological production of natural zeaxanthin is favored due to its safety, potential large-scale production and consumers' preference for natural additives. In this chapter, we describe a rapid screening method based on 16S rRNA gene sequencing and effective HPLC with diode array detector/MS methods for the isolation and identification of zeaxanthin-producing bacteria and their carotenoid analysis.

Screening and profiling of natural ketocarotenoids from environmental aquatic bacterial isolates.

Ketocarotenoids are high-value natural pigments. The red diketocarotenoid astaxanthin particularly exhibits an extraordinary antioxidant activity, which raises its market demand for foods and nutraceuticals. We screened for ketocarotenoid-producing bacteria from both marine and freshwater environments. Phylogenetic analysis, based on 16S rRNA gene sequence, revealed 37 potential producers of ketocarotenoids that are related to α-proteobacteria, comprising 32 strains of Brevundimonas and 5 strains of Erythrobacter. Carotenoids analysis by HPLC-DAD and HPLC-MS revealed two groups; astaxanthin-producers (28 Brevundimonas strains) and adonixanthin-producers (Five Brevundimonas and 5 Erythrobacter strains). Strain FrW-Asx16 exhibited the highest carotenoid production (1060 µg g-1 dry cells with 16.6% astaxanthin). Strain FrW-Asx-5 producing 946.1 µg g-1 dry cells carotenoid exhibited the highest astaxanthin content (∼46%). The most intriguing result is the potential of producing natural colorants from freshwater bacterial isolates, and with high productivity and selectivity, suggesting a great promise for their application in food.

Antibiotics in microbial coculture.

Today, the frequency of discovery of new antibiotics in microbial culture is significantly decreasing. The evidence from whole-genome surveys suggests that many genes involved in the synthesis of unknown metabolites do exist but are not expressed under conventional cultivation conditions. Therefore, it is urgently necessary to study the conditions that make otherwise silent genes active in microbes. Here we overview the knowledge on the antibiotic production promoted by cocultivation of multiple microbial strains. Accumulating evidence indicates that cocultivation can be an effective way to stimulate the production of substances that are not formed during pure cultivation. Characterization of the promotive factors produced by stimulator strains is expected to give clues to the development of effective cultivation conditions for drug discovery.

An ABC transporter involved in the control of streptomycin production in Streptomyces griseus.

We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR, the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb BamHI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus.

Streptomyces metabolites in divergent microbial interactions.

Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites. The diverged action of negative and positive influences onto the physiology of various microorganisms infers the occurrence of complex microbial interactions due to the effect of small molecules produced by Streptomyces. The interactions may serve as a basis for the constitution of biological community.

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.

Analysis of the tryptophanase expression in Symbiobacterium thermophilum in a coculture with Geobacillus stearothermophilus.

The tryptophanase-positive Symbiobacterium thermophilum is a free-living syntrophic bacterium that grows effectively in a coculture with Geobacillus stearothermophilus. Our studies have shown that S. thermophilum growth depends on the high CO2 and low O2 condition established by the precedent growth of G. stearothermophilus. The use of an anoxic atmosphere containing high CO2 allows S. thermophilum to grow independently of G. stearothermophilus, but the cellular yield is ten times lower than that achieved in the coculture. In this study, we characterized the coculture-dependent expression and activity of tryptophanase in S. thermophilum. S. thermophilum cells accumulated a marked amount of indole in a coculture with G. stearothermophilus, but not in the bacterium's pure culture irrespective of the addition of tryptophan. S. thermophilum cells accumulated indole in its pure culture consisting of conditioned medium (medium supplied with culture supernatant of G. stearothermophilus). Proteomic analysis identified the protein specifically produced in the S. thermophilum cells grown in conditioned medium, which was a tryptophanase encoded by tna2 (STH439). An attempt to isolate the tryptophanase-inducing component from the culture supernatant of G. stearothermophilus was unsuccessful, but we did discover that the indole accumulation occurs when 10 mM bicarbonate is added to the medium. RT-PCR analysis showed that the addition of bicarbonate stimulated transcription of tna2. The transcriptional start site, identified within the tna2 promoter, was preceded by the -24 and -12 consensus sequences specified by an alternative sigma factor, σ(54). The evidence suggests that the transcription of some genes involved in amino acid metabolism is σ(54)-dependent, and that a bacterial enhancer-binding protein containing a PAS domain controls the transcription under the presence of high levels of bicarbonate.

Description of Symbiobacterium ostreiconchae sp. nov., Symbiobacterium turbinis sp. nov. and Symbiobacterium terraclitae sp. nov., isolated from shellfish, emended description of the genus Symbiobacterium and proposal of Symbiobacteriaceae fam. nov.

Three novel moderately anaerobic, thermophilic, rod-shaped bacterial strains, KY38(T), KY46(T) and KA13(T), were isolated from shellfish collected on the Pacific coastline of Enoshima, Japan. Phylogenetic analysis of the 16S rRNA gene sequences indicated that these bacteria belong to the genus Symbiobacterium, sharing sequence similarities of 97.8% (KY38(T)), 96.4% (KY46(T)) and 93.3% (KA13(T)) with the type strain of Symbiobacterium thermophilum, the only species of the genus with a validly published name. These isolates reduced nitrate and grew optimally at 55-60 °C. Strains KY38(T) and KA13(T) formed endospore-like structures in the terminal or subterminal part of their cells at low frequencies. Genomic DNA G+C contents were 68.8 (KY38(T)), 67.2 (KY46(T)) and 67.1 (KA13(T)) mol%. The isolates all presented the predominant menaquinone MK-6, major fatty acids iso-C15:0, C16:0 and iso-C17:0 and the major polar lipids phosphatidylglycerol, phosphatidylethanolamine and unknown glycol-containing phospholipids. On the basis of their morphological, physiological and phylogenetic properties, strains KY38(T), KY46(T) and KA13(T) represent three novel species, for which the names Symbiobacterium ostreiconchae sp. nov. (type strain KY38(T) = DSM 27624(T) = KCTC 4567(T) = JCM 15048(T)), Symbiobacterium turbinis sp. nov. (type strain KY46(T) = DSM 27625(T) = KCTC 4568(T) = JCM 15996(T)) and Symbiobacterium terraclitae sp. nov. (type strain KA13(T) = DSM 27138(T) = KCTC 4569(T) = JCM 15997(T)) are proposed. An emended description of the genus Symbiobacterium is also presented. The phylogenetic distinctiveness of the genus Symbiobacterium indicates its affiliation with a novel family, for which the name Symbiobacteriaceae fam. nov. is proposed.

Preparation of optically-active 3-pyrrolidinol and its derivatives from 1-benzoylpyrrolidinol by hydroxylation with Aspergillus sp. and stereo-selective esterification by a commercial lipase.

An effective preparation scheme for optically-active 3-pyrrolidinol and its derivatives based on biological transformation is proposed. Aspergillus sp. NBRC 109513 hydroxylated 1-benzoylpyrrolidine, yielding (S)-1-benzoyl-3-pyrrolidinol with 66 % ee. Kinetic resolution of 1-benzoyl-3-pyrrolidinol by Amano PS-IM lipase formed optically-active 1-benzoyl-3-pyrrolidinol with >99 % ee. (S)-1-Benzoyl-3-pyrrolidinol was successfully converted to 3-pyrrolidinol and its derivatives with by chemical reactions (>99 % ee).

Amide-transforming activity of Streptomyces: possible application to the formation of hydroxy amides and aminoalcohols.

To develop an efficient bioconversion process for amides, we screened our collection of Streptomyces strains, mostly obtained from soil, for effective transformers. Five strains, including the SY007 (NBRC 109343) and SY435 (NBRC 109344) of Streptomyces sp., exhibited marked conversion activities from the approximately 700 strains analyzed. These strains transformed diverse amide compounds such as N-acetyltetrahydroquinoline, N-benzoylpyrrolidine, and N-benzoylpiperidine into alcohols or N,O-acetals with high activity and regioselectivity. N,O-acetal was transformed into alcohol by serial tautomerization and reduction reactions. As such, Streptomyces spp. can potentially be used for the efficient preparation of hydroxy amides and aminoalcohols.

Divergent effects of desferrioxamine on bacterial growth and characteristics.

Desferrioxamines (DF's) are siderophores produced by some groups of bacteria. Previously, we discovered that DFE, produced by Streptomyces griseus, induced divergent developmental phenotypes in various Streptomyces isolates. In this study, we isolated bacteria whose phenotype was affected by the presence of 0.1 mM DFB from soil samples, and studied their phylogenetic position via 16 S rRNA gene-based analysis. Isolates belonging to Microbacterium grew only in the presence of DFB in the medium. DFB promoted growth of some isolates, while significantly inhibiting that of other divergent bacteria. Different groups of isolates were affected, not because of growth-related changes, but because of changes in the colony morphology based on possible stimulation of motility. An isolate affiliated with Janthinobacterium was stimulated for violacein production as well as for pilus formation. The wide and divergent effects of DFB suggest that availability of siderophores significantly affect the structure of microbial community.

Dispensabilities of carbonic anhydrase in proteobacteria.

Carbonic anhydrase (CA) (E.C. 4.2.1.1) is a ubiquitous enzyme catalysing interconversion between CO(2) and bicarbonate. The irregular distribution of the phylogenetically distinct classes of CA in procaryotic genome suggests its complex evolutionary history in procaryotes. Genetic evidence regarding the dispensability of CA under high-CO(2) air in some model organisms indicates that CA-deficient microorganisms can persist in the natural environment by choosing high-CO(2) niches. In this study, we studied the distribution of CA in the genome of Proteobacteria. While a large majority of the genome-sequenced Proteobacteria retained a CA gene(s), intracellular bacterial genera such as Buchnera and Rickettsia contained CA-defective strains. Comparison between CA-retaining and CA- deficient genomes showed the absence of whole coding sequence in some strains and the presence of frameshifted coding sequence in other strains. The evidence suggests that CA is inactivated and lost in some proteobacteria during the course of evolution based on its dispensability.

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.

Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces.

Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu(2+) . Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster 'sco' (the S treptomycescopper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu(2+) into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose-containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two- to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO(4) , suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N'-dimethyl-p-phenylenediamine sulfate. Addition of 10 µM CuSO(4) repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3' terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low-copper condition and for the activation of the multiple Cu(2+) -containing oxidases that play divergent roles in the complex physiology of Streptomyces.

Whole-genome comparison clarifies close phylogenetic relationships between the phyla Dictyoglomi and Thermotogae.

The anaerobic thermophilic bacterial genus Dictyoglomus is characterized by the ability to produce useful enzymes such as amylase, mannanase, and xylanase. Despite the significance, the phylogenetic position of Dictyoglomus has not yet been clarified, since it exhibits ambiguous phylogenetic positions in a single gene sequence comparison-based analysis. The number of substitutions at the diverging point of Dictyoglomus is insufficient to show the relationships in a single gene comparison-based analysis. Hence, we studied its evolutionary trait based on whole-genome comparison. Both gene content and orthologous protein sequence comparisons indicated that Dictyoglomus is most closely related to the phylum Thermotogae and it forms a monophyletic group with Coprothermobacter proteolyticus (a constituent of the phylum Firmicutes) and Thermotogae. Our findings indicate that C. proteolyticus does not belong to the phylum Firmicutes and that the phylum Dictyoglomi is not closely related to either the phylum Firmicutes or Synergistetes but to the phylum Thermotogae.