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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.

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.

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-17

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.

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.

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.

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.

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.

Three novel moderately anaerobic, thermophilic, rod-shaped bacterial strains, KY38T, KY46Tand KA13T, 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 genusSymbiobacterium, sharing sequence similarities of 97.8 % (KY38T), 96.4 % (KY46T) and 93.3 % (KA13T) with the type strain ofSymbiobacterium thermophilum, the only species of the genus with a validly published name. These isolates reduced nitrate and grew optimally at 55–60 °C. Strains KY38Tand KA13Tformed endospore-like structures in the terminal or subterminal part of their cells at low frequencies. Genomic DNA G+C contents were 68.8 (KY38T), 67.2 (KY46T) and 67.1 (KA13T) mol%. The isolates all presented the predominant menaquinone MK-6, major fatty acids iso-C15 : 0, C16 : 0and iso-C17 : 0and the major polar lipids phosphatidylglycerol, phosphatidylethanolamine and unknown glycol-containing phospholipids. On the basis of their morphological, physiological and phylogenetic properties, strains KY38T, KY46Tand KA13Trepresent three novel species, for which the namesSymbiobacterium ostreiconchaesp. nov. (type strain KY38T = DSM 27624T = KCTC 4567T = JCM 15048T),Symbiobacterium turbinissp. nov. (type strain KY46T = DSM 27625T = KCTC 4568T = JCM 15996T) andSymbiobacterium terraclitaesp. nov. (type strain KA13T = DSM 27138T = KCTC 4569T = JCM 15997T) are proposed. An emended description of the genusSymbiobacteriumis also presented. The phylogenetic distinctiveness of the genusSymbiobacteriumindicates its affiliation with a novel family, for which the nameSymbiobacteriaceaefam. nov. is proposed.

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.

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.

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.

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 Bam HI 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 ( r egulation of s treptomycin p roduction). 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 three-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 .

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.

The conservon (cvn) of Streptomyces species encodes a putative membrane-associated signaling complex resembling the eukaryotic G-protein-coupled receptor (GPCR) system. The cvn is widely distributed in the genomes of Actinobacteria, indicating that it plays an important role in this group of bacterial species; however, the exact role of this regulatory system is hitherto poorly understood. In the present study, we generated null mutants for all 13 copies of the cvn operon distributed in the genome of Streptomyces coelicolor A3(2) and observed that the aerial mycelium formation and antibiotic production in a cvn1 mutant were markedly impaired. The cvn1 mutant formed aerial mycelium and produced actinorhodin and undecylprodigiosin at remarkably low levels on solid medium containing 1-2% glucose and at high levels on medium containing 6-10% glucose. The same phenotype as this was observed with a cvnA1 mutant. Transcriptional analyses revealed that the expression of sigU encoding a vegetative sigma factor was upregulated in the cvn1 mutant. Overexpression of rsuA encoding the σ(SigU) antagonist restored aerial mycelium formation and pigment production in the cvn1 mutant, suggesting that the developmental defect in the cvn1 mutant is based on the high expression level of sigU.

Stress-response sigma factor σH is negatively regulated by its cognate anti-sigma factor RshA in Streptomyces griseus. As the overexpression of RshA in the wild-type strain confers a distinctive bald phenotype (deficiency in aerial mycelium formation and streptomycin production), RshA is supposed to associate with not only σH but also another regulatory element that plays a crucial role in the developmental control of S. griseus. Here, we show that an anti-sigma factor antagonist BldG associates with RshA and negatively regulates its activity. The bald phenotype conferred by the overexpression of rshA was restored to the wild-type phenotype by the coexpression with bldG. The in vivo and in vitro protein interaction analyses demonstrated the specific association between RshA and BldG. A bldG mutant exhibited a distinctive bald phenotype and was defective in the σH-dependent transcription activities. The positive regulatory role of BldG regarding the σH activity was verified by an in vitro transcriptional analysis, in which the inhibition of σH-dependent transcription by RshA was abolished by the addition of BldG in a dose-responsive manner. Overall, evidence suggests that BldG serves as a master switch for both stress-response and developmental gene expression based on its association with multiple anti-sigma factors in S. griseus.

Widespread interspecific stimulation of antibiotic production occurs in strains of Streptomyces owing to the activity of diffusible substances, as previously determined in our investigations of the cross-feeding effect. In this study, we newly isolated a substance produced by a Streptomyces strain closely related to Streptomyces scabrisporus, based on the observation that this substance induced the production of an unknown antibiotic in another strain related to Streptomyces griseorubiginosus. This substance, named promomycin, is a polyether structurally related to lonomycin. Promomycin itself had an antibiotic activity, but it stimulated antibiotic production in multiple Streptomyces strains at sub-inhibitory concentrations. Evidence implies that this stimulation effect is widespread within this group of bacteria.

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).

We cloned a DNA fragment that suppressed the aerial-mycelium-deficient phenotype in an amfS mutant of Streptomyces griseus when it was introduced into the cells via a high-copy-number plasmid. The sasABCDR gene cluster was identified as being responsible for this suppressive activity. The proteins encoded by sasABCD were of unknown function, but the operon structure was found to be conserved in all the strains of Streptomyces spp. and related organisms whose genomes have been sequenced. sasR, the flanking opposite coding sequence, encoded a putative DNA-binding protein. Subcloning revealed that the presence of all five coding sequences was essential for complete suppression. Scanning electron microscopy of Streptomyces griseus strains carrying the sas gene cluster at a high copy-number revealed that bundle-like structures consisting of several aerial hyphae were often formed. S1 nuclease protection analyses were performed to identify the transcriptional start site in the promoters preceding sasA and sasR. The promoter preceding sasA was highly active during vegetative growth. Null mutants for sasABCD among the S. griseus and S. coelicolor A3(2) cells exhibited bald phenotypes; this suggested a positive regulatory role of this gene cluster in the onset of morphogenesis in these two phylogenetically distinct Streptomyces species.

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.

Aims: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. Methods and Results: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25°C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. Conclusion: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. Significance and Impact of the Study: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.

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.

Symbiobacterium thermophilum is a thermophilic bacterium that was originally isolated by its ability to produce a thermostable tryptophanase from compost collected at Hiroshima, Japan (19). This bacterium is characterized by a syntrophic property that is reproduced under a laboratory culture condition; although it does not grow or shows impaired growth with a very low cellular yield in pure culture, it effectively propagates, achieving a density of approximately 5 × 108 cells/ml, when it is cocultured with the cognate Bacillus sp. strain S, which was isolated together with S. thermophilum (19). The molecular phylogenetic and genomic sequencing studies (13, 22) uncovered unique genetic and taxonomic features of S. thermophilum. S. thermophilum exhibits a number of genetic features associated with Firmicutes (low-G+C, gram-positive bacteria represented by Bacillus and Clostridium), despite its high G+C content (68.5%). This unusual property has now become a new topic in bacterial systematics (2, 4) and suggests that this kind of bacterium has been left uncharacterized due to its “unculturable nature,” despite its wide distribution in the natural environment (20). We are interested in the fundamental details of the syntrophic feature of S. thermophilum and expect that this information will provide us with new knowledge regarding not only the microbial physiology but also the issue of unculturability of environmental microorganisms. Recently, we discovered that a pure culture of S. thermophilum grew effectively under high atmospheric CO2 concentrations (24). The lines of evidence suggest that the organism's dependence on CO2 for growth is based on the requirement for bicarbonate, which is required for the reaction catalyzed by essential enzymes such as phosphoenolpyruvate carboxylase and acetyl-coenzyme A carboxylase. The requirement for exogenous bicarbonate can be attributed to the lack of carbonic anhydrase, which catalyzes the conversion of CO2 to bicarbonate (17). While bacteria that retain carbonic anhydrase can obtain sufficient bicarbonate for growth by catalytic conversion from environmental CO2, those lacking this enzyme depend on the high concentration of bicarbonate that naturally occurs in the environment. In fact, knockout mutants for a carbonic anhydrase gene confer a CO2 dependence phenotype in Ralstonia eutropha (8) and Escherichia coli (10). The result of genomic sequencing revealed that S. thermophilum does not retain the gene for carbonic anhydrase (22). Based on these observations, we speculate that S. thermophilum grows by using the CO2 that is generated by the cognate Bacillus sp. strain S. Although a supply of CO2 markedly promoted the growth of S. thermophilum, it was insufficient to fully explain its dependence on coculture with Bacillus for growth. The cellular yield of S. thermophilum obtained in the pure culture under conditions of high atmospheric CO2 concentrations was approximately 1 × 107 cells/ml, which corresponds to less than 10% of that achieved by coculture with Bacillus strain S (12, 24). It suggested that the Bacillus strain benefits S. thermophilum not only by supplying CO2 but also by supplying (or eliminating) some other metabolite(s). In this study, based on the observation that dialysis culture promoted the growth of pure S. thermophilum in cultures, we assessed the possibility that the Bacillus strain removes or inactivates a self-growth inhibitor produced by S. thermophilum. We isolated substances from pure cultures of S. thermophilum that inhibited its growth and identified them as indole alkaloids, which have been known as antibacterial metabolites in several gram-negative bacteria. Although the inhibitory activities of these substances do not fully explain the occurrence of self-growth inhibition in S. thermophilum, the evidence strongly suggests that inactivation of the self-growth inhibitor is an important basis of the effective proliferation of S. thermophilum in coculture with Bacillus strain S.

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a cognate Bacillus sp. We have been studying the unique features of S. thermophilum in terms of taxonomy, ecology, genome biology, and physiology. Here we overview current knowledge of this bacterium. Although S. thermophilum shows several physiological properties of Gram-negative bacteria, 16S rRNA gene-based phylogeny indicated that it represents a distinct lineage of Gram-positive bacteria with deep branching between the clades for the high-G+C (Actinobacteria) and the low-G+C (Firmicutes) groups. Ecological study has revealed that S. thermophilum and its relatives are widely distributed in the natural environment, including soil, animal intestines and seawater. A whole genome sequencing study uncovered its unusual features, which overall indicate that this bacterium is a member of Firmicutes despite of its high G+C content (68.7%). The genome appeared to retain fully the genes for primary metabolism, except for carbonic anhydrase. We discovered that carbon dioxide is a marked inducer of the mono-growth of S. thermophilum, and speculated that this is due to a lack of carbonic anhydrase. The lines of evidence suggest that S. thermophilum requires additional conditions for full growth, including not only the supply of an unknown positive factor but also the elimination of oxygen and self-growth inhibitory substances. We conclude that the role of the cognate Bacillus is to establish a complex environment suitable for the growth of S. thermophilum, which is achieved by supplying and removing multiple factors. Understandings of this type of mutualism should provide new insight into microbial physiology as well as the issue of uncultivability.

Streptomyces griseus, a well-known industrial producer of streptomycin, is a member of the genus Streptomyces, which shows a complex life cycle resembling that of fungi. A-factor, a C13 γ-butyrolactone compound, was discovered as a self-regulatory factor or a bacterial hormone to induce morphological differentiation and production of secondary metabolites, including streptomycin, in this organism. Accumulating evidence has revealed an A-factor-triggered signal cascade, which is composed of several key steps or components. These include: (i) AfsA catalyzing a crucial step of A-factor biosynthesis, (ii) the A-factor-specific receptor (ArpA), which acts as a transcriptional repressor for adpA, (iii) adpA, a sole target of ArpA, which encodes a global transcriptional activator AdpA, and (iv) a variety of members of the AdpA regulon, a set of the genes regulated by AdpA. A-factor is biosynthesized via five reaction steps, in which AfsA catalyzes acyl transfer between a β-ketoacyl-acyl carrier protein and the hydroxyl group of dihydroxyacetone phosphate. The receptor ArpA, belonging to the TetR family, is a homodimer, each subunit of which contains a helix-turn-helix DNA-binding motif and an A-factor-binding pocket. The three-dimensional structure and conformational change upon binding A-factor are elucidated, on the basis of X-ray crystallography of CprB, an ArpA homologue. AdpA, belonging to the AraC/XylS transcriptional activator family, binds operators upstream from the promoters of a variety of the target genes and activates their transcription, thus forming the AdpA regulon. Members of the AdpA regulon includes the pathway-specific transcriptional activator gene strR that activates the whole streptomycin biosynthesis gene cluster, in addition to a number of genes that direct the multiple cellular functions required for cellular differentiation in a concerted manner. A variety of A-factor homologues as well as homologues of afsA/arpA are distributed widely among Streptomyces, indicating the significant role of this type of molecular signaling in the ecosystem and evolutional processes.

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.

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell-cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.

Summary 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 cvnA10 conditionally overproduced actinorhodin and performed precocious aerial growth, while a cvnE9 mutant showed the parental phenotype. Transcription of bldG, 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-γ-S were supplied in the reaction respectively. Similarly, D9 associated with and dissociated from B9 (and C9) when GTP and GTP-γ-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.

An ORF encoding the gene (nahA)-related carbohydrate hydrolase was found in chromosomal DNA of the symbiotic thermophile, Symbiobacterium thermophilum. BLASTX results indicated that the product of this structural gene is β-glucosidase or β-N-acetylhexosaminidase. To investigate details of the nahA gene product, cloning of the gene, overproduction of the gene product in Escherichia coli, and purification and characterization of the resulting protein were conducted. The nahA gene was amplified by PCR using fragmented chromosomal DNA of S. thermophilum as a template, sequenced, and then ligated into the BamHI and NdeI sites of plasmid pET-25b(+) to construct the expression vector pST-BNAH-A for overproduction of the gene product. Results of studies on the hydrolytic activity of cell-free extracts against pNP-β-Glc, pNP-β-GlcNAc and pNP-β-GalNAc, obtained by disruption of cultured E. coli cells harboring pST-BNAH-A, suggested that the nahA gene product was β-N-acetylhexosaminidase. Comparison of the amino acid sequence of the recombinant protein with those of other β-N-acetylhexosaminidases indicated that the β-N-acetylhexosaminidase of S. thermophilum is a member of the 3-glycoside hydrolase family. The recombinant enzyme was purified to homogeneity from cell-free extract in an overall yield of 24%. This purified β-N-acetylhexosaminidase possessed thermostability, was stable in alkaline solution, and exhibited greater hydrolytic activity against chitin oligosaccharides than against pNP-β-GlcNAc.

The authors previously reported that interspecific stimulatory events between Streptomyces species for antibiotic production and/or morphological differentiation mediated by putative diffusible metabolites take place at a high frequency. This paper reports the isolation and characterization of a substance produced by Streptomyces griseus that stimulates the growth and development of Streptomyces tanashiensis. The substance was purified from the culture supernatant of S. griseus by using anion-exchange chromatography, gel filtration chromatography and reverse-phase HPLC. FAB-MS and NMR analyses of the purified preparation indicated the substance to be desferrioxamine E (synonym: nocardamine), a siderophore that is widely produced by Streptomyces species and related organisms. Similar stimulatory effects on the growth and development of S. tanashiensis were exerted by desferrioxamine E produced by another actinomycete strain, but not by other siderophores tested, including ferrichrome and nocobactin and free ferric ion. An exogenous supply of desferrioxamine E stimulated secondary metabolite formation and/or morphological differentiation in various actinomycete strains. Disruption of the desferrioxamine biosynthesis gene cluster in Streptomyces coelicolor A3(2) abolished the production of desferrioxamine E and the activity to stimulate the growth and differentiation of S. tanashiensis. The S. coelicolor mutant showed impaired growth and development on Bennett's/glucose agar medium, but it was rescued by the exogenous supply of desferrioxamine E. These results indicate that desferrioxamines play an important role in streptomycete physiology. Similar to several pathogenic bacteria and fungi, S. tanashiensis may be defective in the production of siderophores; however, it can utilize the siderophores excreted by other organisms.

In response to stimulants, such as serum, the yeast cells of the opportunistic fungal pathogen Candida albicans form germ tubes, which develop into hyphae. Yvh1p, one of the 29 protein phosphatases encoded in the C. albicans genome, has 45 % identity with the dual-specific phosphatase Yvh1p of the model yeast Saccharomyces cerevisiae. In this study, Yvh1p expression was not observed during the initial step of germ tube formation, although Yvh1p was expressed constitutively in cell cycle progression of yeast or hyphal cells. In an attempt to analyse the function of Yvh1p phosphatase, the complete ORFs of both alleles were deleted by replacement with hph200–URA3–hph200 and ARG4. Although YVH1 has nine single-nucleotide polymorphisms in its coding sequence, both YVH1 alleles were able to complement the YVH1 gene disruptant. The vegetative growth of Δyvh1 was significantly slower than the wild-type. The hyphal growth of Δyvh1 on agar, or in a liquid medium, was also slower than the wild-type because of the delay in nuclear division and septum formation, although germ tube formation was similar between the wild-type and the disruptant. Despite the slow hyphal growth, the expression of several hypha-specific genes in Δyvh1 was not delayed or repressed compared with that of the wild-type. Infection studies using mouse models revealed that the virulence of Δyvh1 was less than that of the wild-type. Thus, YVH1 contributes to normal vegetative yeast or hyphal cell cycle progression and pathogenicity, but not to germ tube formation.

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus . Here we examined the transcriptional control mechanism for the promoter preceding amfT (P amfT ) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT . Low-resolution analysis showed that P amfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The −35 region of P amfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus , which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind P amfT . Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of P amfT activity. An amfT -depleted mutant also showed a bald phenotype but P amfT activity was not affected. Both the bldD -overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control P amfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus .

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (alpha- d-glucopyranosyl-[1,1]-alpha- d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5-1% ammonium sulfate and 0.5-1% CaCO(3). The addition of CaCO(3) adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.

A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobacterium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth. The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compartments to be performed. Using the bioreactor, growth characteristics of S. thermophilum and Bacillus strain S were assessed under two medium conditions. The growth of S. thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity. In medium containing 2% tryptone peptone, S. thermophilum proliferated up to 4x10(7) cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed. On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S. thermophilum in the co-culture (6x10(7) cells/ml), but also allowed limited pure growth independent of Bacillus strain S (1x10(7) cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S. thermophilum supplied by Bacillus strain S. Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant metabolic changes. The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms.

The biological process of dinitrogen (N2) gas formation from fixed nitrogen compounds such as nitrate (NO3−) plays an important role in maintaining homeostasis of the global environment. Bacterial denitrification was long considered the sole biological reaction responsible for this condition, and its systems have been characterized in detail (3, 8, 20). Denitrification physiologically functions as anaerobic respiration in which NO3− is used as the terminal electron acceptor when oxygen (O2) is unavailable. Known bacterial denitrifying systems consist of four steps that successively reduce NO3− to N2 and involve nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as intermediates. Production of the enzymes catalyzing each step is induced by nitrogen oxides and suppressed by O2 (3, 20). During the past decade, denitrifiers have been discovered in a variety of taxa including filamentous fungi (9, 10), yeasts (15), and actinomycetes (1, 11). All of these novel denitrifiers produce N2O as the major denitrification product. Both nitrogen atoms in the N2O product are derived from nitrate (or nitrite) (1, 9-11, 15). The phenomenon is therefore defined as denitrification, since the process should include the formation of an N—N bond (20). This system in several fungi is localized at respiring mitochondria, where it acts as anaerobic respiration, as it does in bacterial systems (5, 13, 16). Another unique feature of the fungal system is the involvement of cytochrome P450 (P450nor) as NO reductase (Nor) (6). Biological processes other than bacterial denitrification evolve N2 from fixed nitrogen compounds, but their molecular mechanisms and physiological significance remain to be elucidated. We identified simultaneous fungal codenitrification and denitrification, in which a hybrid N2 species is formed by combining two nitrogen atoms derived from NO2− and from other nitrogen compounds (10, 14). The denitrifying fungi Fusarium solani and Cylindrocarpon tonkinense evolve hybrid N2 species (10). The fact that the denitrifying fungus Fusarium oxysporum evolves N2O instead of N2 by codenitrification only when a nitrogen compound in addition to NO2− such as azide, salicylhydroxamic acid, or ammonium (NH4+) is available (14) suggests that the mechanisms of this process differ among fungal species. Anammox is a third N2-generating metabolic process that has been identified in the strictly anaerobic chemolithotrophic Planctomycetales (12), in which NH4+ is combined with NO2− to form N2. The actinomycetes form a unique taxon among gram-positive bacteria. Although actinomycetes naturally proliferate in soil and in aqueous environments, little is known about how they accomplish denitrification. Denitrifiers also occur among actinomycetes (1, 11), and the system of Streptomyces thioluteus has been characterized previously (11). All of the actinomycete denitrifiers found in these studies evolve N2O from NO3− or NO2−, and thus, no known actinomycete strains contain a complete denitrifying system that can thoroughly reduce NO3− to N2. The present study continues screening for denitrifying actinomycetes (11) by using a highly sensitive N2 detection system equipped with an isotope mass spectrometer and NO3− labeled with a stable isotope ([15N]NO3−). The results showed that Streptomyces antibioticus B-546 has N2-producing activity and that most of the N2 molecules are formed via intracellular codenitrification that is induced simultaneously with denitrification.

Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N′-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

Site-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-binding and catalysis. Mutations of two amino acid residues, E13 in the S3 subsite and N219 in the S3/S4 subsites, caused marked changes in kinetic parameters for two substrate peptides with different sequences. Further site-directed mutagenesis at E13 suggested that E13 plays a critical role in forming the correct hydrogen bond network around the active center. In the crystal structure of Rhizomucor miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the Oepsilon atom of N219 forms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhibitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolytic activity without significant loss of clotting activity. This mutant may present a promising candidate for a useful milk coagulant.

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.

Carbon source is one of the environmental factors that affects cellular differentiation of Streptomyces. We have identified the craA gene as a putative negative regulator involved in the carbon-source-dependent initiation of cellular differentiation in Streptomyces griseus. Carbon-source-dependent transcriptional repression of craA, which is caused by binding of a putative repressor protein to its promoter region, is proposed to result in the initiation of aerial mycelium formation. The presence of a craA homologue in the chromosome of Streptomyces coelicolor A3(2) implicates the existence of a similar regulatory mechanism in this organism. The repression of craA-promoter activity in glucose media could be alleviated not only by replacing glucose with maltose but also by supplying copper, which suggests that the stimulatory effect of copper on cellular differentiation in Streptomyces is excerted via abolishment of glucose-repression of craA.

To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (PORF3) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. PORF3 activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of PORF3 mediated by a transcriptional repressor may initiate differentiation in S. griseus.