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.

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.

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.

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.

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.

Involvement of CarA/LitR and CRP/FNR family transcriptional regulators in light-induced carotenoid production in Thermus thermophilus.

Members of the CarA/LitR family are MerR-type transcriptional regulators that contain a C-terminal cobalamin-binding domain. They are thought to be involved in light-induced transcriptional regulation in a wide variety of nonphototrophic bacteria. Based on the distribution of this kind of regulator, the current study examined carotenoid production in Thermus thermophilus, and it was found to occur in a light-induced manner. litR and carotenoid and cobalamin biosynthesis genes were all located on the large plasmid of this organism. litR or cobalamin biosynthesis gene knockout mutants were unable to switch off carotenoid production under dark conditions, while a mutant with a mutation in the downstream gene adjacent to litR (TT_P0055), which encodes a CRP/FNR family transcriptional regulator, was unable to produce carotenoids, irrespective of light conditions. Overall, genetic and biochemical evidence indicates that LitR is bound by cobalamin and associates with the intergenic promoter region between litR and crtB (phytoene synthase gene), repressing the bidirectional transcription of litR and crtB. It is probable that derepression of LitR caused by some photodependent mechanism induces the expression of TT_P0055 protein, which serves as a transcriptional activator for the crtB operon and hence causes the expression of carotenoid biosynthesis and the DNA repair system under light condition.

Longispora fulva sp. nov., isolated from a forest soil, and emended description of the genus Longispora.

A novel actinomycete, strain KZ0017(T), was isolated from a forest soil collected in Ohnuma, Fukushima, Japan. Strain KZ0017(T) formed spore chains borne on top of short sporophores arising from vegetative hyphae. Spores were non-motile and cylindrical with smooth surfaces. Strain KZ0017(T) contained meso-diaminopimelic (A(2)pm) acid, 3-OH A(2)pm, d-glutamic acid, glycine and l-alanine in the cell-wall peptidoglycan, and xylose, mannose, galactose, rhamnose and ribose in cell-wall hydrolysates. The acyl type of the cell-wall polysaccharides was glycolyl. The predominant menaquinones were MK-10(H(4)) and MK-10(H(6)); MK-10(H(8)) was a minor component. The polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol and several unknown lipids and glycolipids. The major fatty acids were iso-C(16 : 0), 10-methyl-C(17 : 0) and iso-C(17 : 1)ω9c. The DNA G+C content was 70.7 mol%. The 16S rRNA gene sequence of the isolate formed a monophyletic cluster with the single member of the genus Longispora in the family Micromonosporaceae. On the basis of morphological, chemotaxonomic and phylogenetic properties, strain KZ0017(T) represents a novel species of the genus Longispora, for which the name Longispora fulva sp. nov. is proposed; the type strain is KZ0017(T) ( = NBRC 105670(T) = DSM 45356(T)).

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)).

Isolation of bacteria whose growth is dependent on high levels of CO2 and implications of their potential diversity.

Although some bacteria require an atmosphere with high CO(2) levels for their growth, CO(2) is not generally supplied to conventional screening cultures. Here, we isolated 84 bacterial strains exhibiting high-CO(2) dependence. Their phylogenetic affiliations imply that high-CO(2) culture has potential as an effective method to isolate unknown microorganisms.

Deinococcus misasensis and Deinococcus roseus, novel members of the genus Deinococcus, isolated from a radioactive site in Japan.

Two gamma- and UV-radiation resistant, Gram-positive, red- or pink-pigmented, rod-shaped, strictly aerobic, oxidase- and catalase-positive bacterial strains, TDMA-25T and TDMA-uv51T, were isolated from fresh water collected at Misasa, a radioactive site in Japan. Phylogenetic analysis based on 16S rRNA gene sequences placed both in a distinct lineage in the family Deinococcaceae, and the highest degrees of sequence similarity determined belonged to Deinococcus maricopensis LB-34T (88.8-89.3%), Deinococcus pimensis KR-235T (86.4-86.7%) and Deinococcus yavapaiensis KR-236T (86.1%). The DNA G+C content of the strains was 53-58 mol%. The major respiratory quinone was MK-8. The predominant fatty acids were C15:0 iso, C16:0 iso, C13:0 iso, C17:0 iso, C16:0, C13:0 anteiso, C15:0 and C12:0 iso. The strains degraded gelatin, casein, starch and Tween 80. Unique physiological characteristics, differences in their fatty acid profiles, and genotypic and phylogenetic features, differentiated strains TDMA-25T and TDMA-uv51T from closely related Deinococcus species. Hence, the two strains are described as novel species of the genus Deinococcus. The names Deinococcus misasensis sp. nov. (type strain TDMA-25T=JCM 14369=NBRC 102116=CCUG 53610) and Deinococcus roseus sp. nov. (type strain TDMA-uv51T=JCM 14370=NBRC 102117=CCUG 53611) are proposed.

Characterization of developmental colony formation in Corynebacterium glutamicum.

We report that Corynebacterium glutamicum colonies exhibit a developmental transition in culture. When cultured on a routinely used complete medium (CM2B), this bacterium first formed a flat translucent colony. Subsequently, some parts of this colony expanded to form small spherical yellow colonies that finally developed into a single large yellow colony. The small flat colony consisted of long thick cells, which were occasionally V or Y shaped, while the large yellow colony consisted of short small rods. A similar colony development pattern was observed in Corynebacterium ammoniagenes and Corynebacterium callunae. Analysis following shotgun cloning revealed that the introduction of a multi-copy-number plasmid carrying amtR, a global transcriptional regulator for nitrogen metabolism, into C. glutamicum cells induced precocious colony development. An amtR-null C. glutamicum mutant exhibited delayed development. Detailed observations of C. glutamicum cells cultured on CM2B medium containing buffers at various pH values revealed that the colony growth was rapid at a pH value of 6.4 or higher and slow but distinct at a pH of less than 6.4. This pH threshold increased to 6.8 following the addition of 0.1% glucose into the medium.

Distribution of Symbiobacterium thermophilum and related bacteria in the marine environment.

We study the ecological distribution of a unique syntrophic bacterium, Symbiobacterium thermophilum, and related bacteria. In this study, we found that they were frequently obtained from seashells and several marine samples. Symbiobacterium also grew from sterilized oyster shells incubated undersea for 2 or 3 months on the coast of Shimoda, Shizuoka, Japan. 16S rRNA gene-based phylogeny of the clones obtained from the Symbiobacterium-positive cultures demonstrated the potential diversity of this bacterial group, which constitutes a distinct clade between Actinobacteria and Firmicutes. We successfully isolated two new Symbiobacterium strains from oyster shells. 16S rRNA gene-based phylogeny indicated that one belongs to S. thermophilum, and that the other is affiliated with a different species. We also isolated Ureibacillius spp., which showed activity supporting the growth of S. thermophilum.

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.

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.

Role of sigmaH paralogs in intracellular melanin formation and spore development in Streptomyces griseus.

Streptomyces griseus possesses multiple stress-response sigma factors including sigma(H). Previously, we have suggested that sigma(H) and related sigma factors are involved in the developmental control of S. griseus. Herein, we studied the role of two sigma(H) paralogs--sigma(F) and sigma(N)--which are encoded in tandem coding sequences of sigF-sigN in S. griseus [sigma(N) has been described as sigma(L) previously (Gene 320:127, 2003)]. A sigF mutant produced decreased levels of intracellular melanin and formed irregular spores. A triple mutant for sigHNF exhibited defective melanin production. While sigN was transcribed by three tandem promoters during the early to late growth phases, sigF was transcribed in the late developmental phase by a single promoter. The activity of the promoter preceding the rpp operon (Prpp), which is responsible for the intracellular melanin biosynthesis, was decreased in the sigF mutant and abolished in the sigHNF, adpA and A-factor biosynthesis mutants. The in vitro transcription assay demonstrated that Esigma(F) transcribed the rpp promoter. Both Esigma(F) and Esigma(N) transcribed a sigma(H)-dependent promoter that preceded the sigH operon, and their activities were repressed by the addition of RshA, an anti-sigma(H) protein. Overall, the results suggest that the three sigma factors have similar functions and that they are required for spore development and pigmentation. The transcription of the rpp operon is regulated both by the stress-response sigma factors and the A-factor regulatory cascade.