Search

Streptomyces griseus mutants exhibiting deficient glucose repression of [beta]-galactosidase activity on lactosecontaining minimal medium supplemented with a high concentration of glucose were isolated. One of these mutants had a 12-bp deletion in cebR, which encodes a LacI/GalR family regulator. Disruption of cebR in the wild-type strain caused the same phenotype as the mutant, indicating that CebR is required for glucose repression of [beta]-galactosidase activity. Recombinant CebR protein bound to a 14-bp inverted-repeat sequence (designated the CebR box) present in the promoter regions of cebR and the putative cellobiose utilization operon, cebEFG-bglC. The DNA-binding activity of CebR was impaired by cellooligosaccharides, including cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. In agreement with this observation, transcription from the cebE and cebR promoters was greatly enhanced by the addition of cellobiose to the medium. Seven other genes containing one or two CebR boxes in their upstream regions were found in the S. griseus genome. Five of these genes encode putative secreted proteins: two cellulases, a cellulose-binding protein, a pectate lyase, and a protein of unknown function. These five genes and cebEFG-bglC were transcribed at levels 4 to 130 times higher in the AcebR mutant than in the wild-type strain, as determined by quantitative reverse transcription-PCR. These findings indicate that CebR is a master regulator of cellulose/cellooligosaccharide catabolism. Unexpectedly, the AcebR mutant formed very few aerial hyphae on lactose-containing medium, demonstrating a link between carbon source utilization and morphological development. doi: 10.1128/JB.00703-09

The GinI/GinR quorum-sensing system represses oxidative fermentation, including acetic acid and gluconic acid fermentation, as well as antifoam activity in Gluconacetobacter intermedius NCI1051. An 89 aa protein, GinA, whose production is induced by the quorum-sensing system, represses both oxidative fermentation and antifoam activity via a still unknown mechanism, although an OmpA family protein, GmpA, as a target of the GinI/GinR quorum-sensing system via GinA, has been found to repress oxidative fermentation. In this study, four novel GinA-inducible genes (gltA, pdeA, pdeB and nagA) were identified and their involvement in oxidative fermentation and antifoam activity was examined by gene disruption. Disruption of nagA (which encodes a putative N-acetylglucosamine-6-phosphate deacetylase) decreased the growth rate in the exponential growth phase, indicating that nagA was required for the rapid growth of the strain. This unexpected finding revealed a new aspect of the GinI/GinR quorum-sensing system: it accelerates exponential growth by induction of nagA. In contrast, gltA (a putative glycosyltransferase) and pdeA (a putative cyclic-di-GMP phosphodiesterase) were shown to repress oxidative fermentation, including acetic acid and gluconic acid fermentation, gltA was also shown to repress antifoam activity. Disruption of pdeB (a putative phosphodiesterase/diguanylate cyclase) caused no phenotypic changes. Taking our previous results into consideration, these results showed an apparently complex mechanism for repressing oxidative fermentation by the quorum-sensing system; at least three GinA-inducible genes, gltA, pdeA and gmpA, were involved in the repression of oxidative fermentation by the GinI/GinR quorum-sensing system, the most characteristic feature of the acetic acid bacteria.

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.

Small noncoding RNAs (sRNAs) have been shown to control diverse cellular processes in prokaryotes. To identify and characterize novel bacterial sRNAs, a gram-positive, soil-inhabiting, filamentous bacterium, Streptomyces griseus, was examined, on the assumption that Streptomyces should express sRNAs as important regulators of morphological and physiological differentiation. By bioinformatics investigation, 54 sRNA candidates, which were encoded on intergenic regions of the S. griseus chromosome and were highly conserved in those of both Streptomyces coelicolor A3(2) and Streptomyces avermitilis, were selected. Of these 54 sRNA candidates, 17 transcripts were detected by Northern blot analysis of the total RNAs isolated from cells grown on solid medium. Then, the direction of transcription of each sRNA candidate gene was determined by S1 nuclease mapping, followed by exclusion of four sRNA candidates that were considered riboswitches of their downstream open reading frames (ORFs). Finally, a further sRNA candidate was excluded because it was cotranscribed with the upstream ORF determined by reverse transcription-PCR. Thus, 12 sRNAs ranging in size from 40 to 300 nucleotides were identified in S. griseus. Seven of them were apparently transcribed in a growth phase-dependent manner. Furthermore, of the 12 sRNAs, the expression profiles of 7 were significantly influenced by a mutation of adpA, which encodes the central transcriptional regulator of the A-factor regulatory cascade involved in both morphological differentiation and secondary metabolism in S. griseus. However, disruption of all 12 sRNA genes showed no detectable phenotypic changes; all the disruptants grew and formed aerial mycelium and spores with the same time course as the wild-type strain on various media and produced streptomycin similarly to the wild-type strain.

A-factor (2-isocapryloyl-3R-hydroxymethyl-[gamma]-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant [DELTA]afsA. A-factor was added at a concentration of 25 ng [ml.sup.-1] to mutant [DELTA]afsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Curcuminoids, which are produced specifically by plants of the order Zingiberales, have long been used as food additives because of their aromatic, stimulant and colouring properties and as traditional Asian medicines because of their anti-tumour, antioxidant and hepatoprotective activities. Curcuminoids are therefore attractive targets for metabolic engineering. An artificial curcuminoid biosynthetic pathway, including reactions of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate : CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa), a type III polyketide synthase, was constructed in Escherichia coli for the production of curcuminoids. Cultivation of the recombinant E. coli cells in the presence of tyrosine or phenylalanine, or both, led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane. Another E. coli system carrying 4CL and CUS genes was also used for high-yield production of curcuminoids from exogenously supplemented phenylpropanoid acids: p-coumaric acid, cinnamic acid and ferulic acid. The yields of curucminoids were up to ~100 mg [l.sup.-1]. Furthermore, this system gave approximately 60 mg curcumin [l.sup.-1] from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.

We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G + C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5'-GGA-3', are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a [gamma]-butyrolactone signaling molecule) regulatory cascade in S. griseus.

A number of gram-negative bacteria regulate gene expression in a cell density-dependent manner by quorum sensing via N-acylhomoserine lactones (AHLs). Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, produces three different AHLs, N-decanoyl-L-homoserine lactone, N-dodecanoyl-L-homoserine lactone, and an N-dodecanoyl-L-homoserine lactone with a single unsaturated bond in its acyl chain, as determined by liquid chromatography-tandem mass spectrometry. Two genes encoding an AHL synthase and a cognate regulator were cloned from strain NCI1051 and designated ginI and ginR, respectively. Disruption of ginI or ginR abolished AHL production, indicating that NCI1051 contains a single set of quorum-sensing genes. Transcriptional analysis showed that ginI is activated by GinR, which is consistent with the finding that there is an inverted repeat whose nucleotide sequence is similar to the sequence bound by members of the LuxR family at position -45 with respect to the transcriptional start site of ginI. A single gene, designated ginA, located just downstream of ginI is transcribed by read-through from the GinR-inducible ginI promoter. A ginA mutant, as well as the ginI and ginR mutants, grew more rapidly in medium containing 2% (vol/vol) ethanol and accumulated acetic acid at a higher rate with a greater final yield than parental strain NCI1051. In addition, these mutants produced larger amounts of gluconic acid than the parental strain. These data demonstrate that the GinI/GinR quorum-sensing system in G. intermedius controls the expression of ginA, which in turn represses oxidative fermentation, including acetic acid and gluconic acid fermentation.

AtrA, a transcriptional activator for actII-ORF4, encoding the pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2), has been shown to bind the region upstream from the promoter of strR, encoding the pathway-specific transcriptional activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus [Uguru et al. (2005) Mol Microbiol 58, 131-150]. The atrA orthologue (atrA-g) in S. griseus was constitutively transcribed throughout growth from a promoter located about 250 nt upstream of the translational start codon, as determined by S1 nuclease mapping. DNase I footprinting showed that histidine-tagged AtrA-g bound an inverted repeat located upstream of strR at positions -117 to -142 relative to the transcriptional start point of strR as +1. This AtrA-g-binding site was between two AdpA-binding sites at approximately nucleotide positions -270 and -50. AdpA is a central transcriptional activator in the A-factor regulatory cascade and essential for the transcription of strR. AtrA-g and AdpA simultaneously bound the respective binding sites. In contrast to AdpA, AtrA-g was non-essential for strR transcription; an atrA-g-disrupted strain produced streptomycin on routine agar media to the same extent as the wild-type strain. However, the atrA-g-disrupted strain tended to produce a smaller amount of streptomycin than the wild-type strain under some conditions, for example, on Bennett agar containing 1% maltose and on a minimal medium. Therefore, AtrA-g had a conditionally positive effect on streptomycin production, as a tuner, probably by enhancing the AdpA-dependent transcriptional activation of strR in a still unknown manner.

Grixazone (GX), which is a diffusible yellow pigment containing a phenoxazinone chromophore, is one of the secondary metabolites under the control of A-factor (2-isocapryloyl-3R-hydroxymethyl-[gamma]-butyrolactone) in Streptomyces griseus. GX production is also induced by phosphate starvation. The whole biosynthesis gene cluster for GX was cloned and characterized. The gene cluster consisting of 13 genes contained six transcriptional units, griT, griSR, griR, griAB, griCDEFG, and griJIH. During cultivation in a phosphate-depleted medium, the six promoters were activated in the order (i) griR, (ii) griC and griJ, and (iii) griT, griS, and griA. Disruption of griR, which encodes a SARP family transcriptional regulator, abolished the transcriptional activation of all other genes in the cluster. In addition, ectopic expression of griR from a constitutively active promoter resulted in GX overproduction even in the absence of AdpA, a key transcriptional activator in the A-factor regulatory cascade, and in the presence of phosphate at a high concentration. GriR monomers bound direct repeat sequences in the griC and griJ promoters in a cooperative manner. Therefore, the early active genes (griCDEFG and griJIH), all of which, except for griG (which encodes a transporter-like protein), encode the GX biosynthesis enzymes, were directly activated by GriR. The transcription of griR was greatly reduced in the presence of phosphate at a high concentration and was hardly detected in the absence of AdpA. These findings showed that both A-factor and phosphate depletion signals were required for griR transcription and both signals were transmitted to the GX biosynthesis genes solely via the griR promoter. doi:10.1128/JB.00055-07

An arylamine N-acetyltransferase (NAT) responsible for the N acetylation of exogenous 3-amino-4-hydroxy-benzoic acid in Streptomyces griseus was identified and characterized. This enzyme was distinct from other eukaryotic and bacterial NATs in that it acetylated various 2-aminophenol derivatives more effectively than it acetylated 5-aminosalicylic acid, and thus it may be involved in the metabolism of xenobiotic compounds.

A factor (2-isocapryloyl-3R-hydroxymethyl-[gamma]-butyrolactone) is a representative of the [gamma]-butyrolactone autoregulators that trigger secondary metabolism and morphogenesis in the Gram-positive, filamentous bacterial genus Streptomyces. Here, we report the A factor biosynthesis pathway in Streptomyces griseus. The monomeric AfsA, containing a tandem repeat domain of [approximately equal to] 80 aa, catalyzed [beta]-ketoacyl transfer from 8-methyl-3-oxononanoyl-acyl carrier protein to the hydroxyl group of dihydroxyacetone phosphate (DHAP), thus producing an 8-methyl-3-oxononanoyI-DHAP ester. The fatty acid ester was nonenzymatically converted to a butenolide phosphate by intramolecular aldol condensation. The butenolide phosphate was then reduced by BprA that was encoded just downstream of afsA. The phosphate group on the resultant butanolide was finally removed by a phosphatase, resulting in formation of A factor. The 8-methyl-3-oxononanoyI-DHAP ester produced by the action of AfsA was also converted to A factor in an alternative way; the phosphate group on the ester was first removed by a phosphatase and the dephosphorylated ester was converted nonenzymatically to a butenolide, which was then reduced by a reductase different from BprA, resulting in A factor. Because introduction of afsA alone into Escherichia coli caused the host to produce a substance having A factor activity, the reductase(s) and phosphatase(s) were not specific to the A factor biosynthesis but commonly present in bacteria. AfsA is thus the key enzyme for the biosynthesis of y-butyrolactones. A factor | Streptomyces griseus | AfsA | cell-to-cell signaling

AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position -70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metailoendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.

Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NAD[P.sup.+] reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahy-droxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter.

AdpA is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for secondary metabolism and morphological differentiation. Of the five chymotrypsin-type serine protease genes, sprA, sprB, and sprD were transcribed in response to AdpA, showing that these protease genes are members of the AdpA reguion. These proteases were predicted to play the same physiological role, since these protease genes were transcribed in a similar time course during growth and the matured enzymes showed high end-to-end similarity to one another. AdpA bound two sites upstream of the sprA promoter approximately at positions -375 and -50 with respect to the transcriptional start point of sprA. Mutational analysis of the AdpA-binding sites showed that both AdpA-binding sites were essential for transcriptional activation. AdpA bound a single site at position -50 in front of the sprB promoter and greatly enhanced the transcription of sprB. The AdpA-binding site at position -40 was essential for transcription of sprD, although there was an additional AdpA-binding site at position -180. Most chymotrypsin activity excreted by S. griseus was attributed to SprA and SprB, because mutant [DELTA]sprAB, having a deletion in both sprA and sprB, lost almost all chymotrypsin activity, as did mutant [DELTA]adpA. Even the double mutant [DELTA]sprAB and triple mutant [DELTA]sprABD grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.

A-factor (2-isocapryloyl-3R-hydroxymethyl-[gamma]-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions -270 and -50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.

AdpA is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus, forming an AdpA regulon. Trypsin-like activity was detected at a late stage of growth in the wild-type strain but not in an A-factor-deficient mutant. Consistent with these observations, two trypsin genes, sprT and sprU, in S. griseus were found to be members of the AdpA regulon; AdpA activated the transcription of both genes by binding to the operators located at about -50 nucleotide positions with respect to the transcriptional start point. The transcription of sprT and sprU, induced by AdpA, was most active at the onset of sporulation. Most trypsin activity exerted by S. griseus was attributed to SprT, because trypsin activity in an sprT-disrupted mutant was greatly reduced but that in an sprU-disrupted mutant was only slightly reduced. This was consistent with the observation that the amount of the sprT mRNA was much greater than that of the sprU transcript. Disruption of both sprT and sprU (mutant [DELTA]sprTU) reduced trypsin activity to almost zero, indicating that no trypsin genes other than these two were present in S. griseus. Even the double mutant [DELTA]sprTU grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.