Production of d-Branched-Chain Amino Acids by Lactic Acid Bacteria Carrying Homologs to Isoleucine 2-Epimerase of Lactobacillus buchneri.

Isoleucine 2-epimerase (ILEP) is a novel branched-chain amino acid racemase isolated from Lactobacillus buchneri. In this study, we examined production of free d-branched-chain amino acids such as d-valine, d-leucine, and d-allo-isoleucine, using lactic acid bacteria carrying homologs to ILEP. Twelve selected strains of lactic acid bacteria were grown at optimal growth temperatures and accumulation of d-branched-chain amino acids in the medium was monitored in exponential, early stationary, and stationary phases. To analyze the d-branched-chain amino acids, enantiomers in the medium were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-isobutyryl-l-cysteine. The resultant fluorescent isoindole derivatives were analyzed on an octadecylsilyl stationary phase using ultra-high performance liquid chromatography. The analyses revealed that the seven following lactic acid bacteria carrying homologs showing 53-60% amino acid sequence identity to the L. buchneri ILEP accumulate d-branched-chain amino acids: Lactobacillus fermentum and Weissella paramesenteroides produce d-valine, d-leucine, and d-allo-isoleucine; Lactobacillus reuteri, Leuconostoc mesenteroides subsp. mesenteroides, and Leuconostoc gelidum subsp. gasicomitatum accumulate d-leucine and d-allo-isoleucine; and Lactobacillus vaginalis and Leuconostoc pseudomesenteroides produce d-allo-isoleucine. These results suggest that d-branched-chain amino acids are produced by a variety of lactic acid bacteria species, particularly those carrying homologs to the ILEP.

Heterologous production of kasugamycin, an aminoglycoside antibiotic from Streptomyces kasugaensis, in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster.

Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50 years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.

Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli.

In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.

Identification of site-specific degradation in bacterially expressed human fibroblast growth factor 4 and generation of an aminoterminally truncated, stable form.

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for tumorigenesis in humans and the development of mammalian embryos. The secreted, mature form of human FGF4 is thought to be comprised of 175 amino acid residues (proline(32) to leucine(206), Pro(32)-Leu(206)). Here, we found that bacterially expressed, 6× histidine (His)-tagged human FGF4 (Pro(32)-Leu(206)) protein, referred to as HishFGF4, was unstable such as in phosphate-buffered saline. In these conditions, site-specific cleavage, including between Ser(54) and Leu(55), in HishFGF4 was identified. In order to generate stable human FGF4 derivatives, a 6× His-tagged human FGF4 (Leu(55)-Leu(206)), termed HishFGF4L, was expressed in Escherichia coli. HishFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HishFGF4L was considered as sufficiently stable. HishFGF4L exerted significant mitogenic activities in mouse embryonic fibroblast Balb/c 3T3 cells. In the presence of PD173074, an FGF receptor inhibitor, the growth-stimulating activity of HishFGF4L disappeared. Taken together, we suggest that HishFGF4L is capable of promoting cell growth via an authentic FGF signaling pathway. Our study provides a simple method for the production of a bioactive human FGF4 derivative in E. coli.

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds.

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro(32) -Leu(206) ) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine-tagged bovine FGF4 (Pro(32) -Leu(206) ), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly(25) -Leu(206) ) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.

Intact structure of EGAM1 homeoproteins and basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C contribute to their nuclear localization in mouse embryonic stem cells.

Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells.

Common amino acid sequences deduced from coding exons of the porcine FGF4 gene in two breeds and production of the encoded protein in Escherichia coli.

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the development of mammalian embryos. Here we identified common amino acid sequences predicted from coding exons of the FGF4 gene in five pigs of two breeds, and HispFGF4, a 6× histidine-tagged porcine FGF4, was produced in Escherichia coli. HispFGF4 was purified efficiently from the supernatant of cell lysate by heparin column chromatography. In a porcine embryonic fibroblast cell line, HispFGF4 showed significant mitogenic activities at concentrations as low as 0.001 nM (p<0.01). To the best of our knowledge, this is the first report describing the complete nucleotide sequence of coding exons for the porcine FGF4 protein in two breeds, together with the production of a recombinant, bioactive porcine FGF4 derivative.

Cloning of dfdA genes from Terrabacter sp. strain DBF63 encoding dibenzofuran 4,4a-dioxygenase and heterologous expression in Streptomyces lividans.

A dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 harbors the dbfA and dbfBC genes for DF degradation and the fln-dbfA, pht, and pca gene clusters for the utilization of fluorene (FN) as a sole carbon source. From this strain, dfdA1, the gene encoding the second DF dioxygenase was detected using degenerate polymerase chain reaction (PCR) and the dfdA1A2A3A4 genes were cloned from a cosmid library of the DBF63 genome. Nucleotide sequencing revealed that the dfdA genes showed considerably high identities with those of other actinobacteria, such as Terrabacter sp. strain YK3 and Rhodococcus sp. strain HA01. In the neighboring region of the dfdA genes, as many as 11 homologs for transposase and integrase genes and the putative extradiol dioxygenase gene disrupted by an insertion sequence (dfdB::ISTesp2) were found, suggesting that repeated gene rearrangement had occurred. Quantitative reverse transcription-PCR analysis revealed that dfdA1 was expressed primarily in the DF-fed strain, whereas dbfA1 was expressed in the FN-cultured strain, apparently indicating that the dfdA genes are induced by DF for the initial hydroxylation of DF in strain DBF63. Furthermore, two polycistronic gene cassettes consisting of either dfdA or dbfA together with the dbfBC gene were constructed and expressed heterologously in Streptomyces lividans, degrading DF to salicylate. Furthermore, the expressed DfdA dioxygenase degraded dibenzo-p-dioxin, carbazole, dibenzothiophene, anthracene, phenanthrene, and biphenyl, thereby exhibiting a broader substrate range than that of the DbfA dioxygenase.

Severe inhibition of in vitro cardiomyogenesis in mouse embryonic stem cells ectopically expressing EGAM1C homeoprotein.

Embryoid bodies were prepared from mouse embryonic stem cells expressing exogenous EGAM1C to analyze their ability to differentiate toward terminally differentiated cell types. The generation of cardiomyocytes was severely suppressed in Egam1c transfectants without upregulation of Nkx2-5, a crucial gene for cardiomyogenesis. These results indicate that EGAM1C is capable of affecting terminal differentiation in mouse embryonic stem cells.

Preferential emergence of cell types expressing markers for primitive endoderm lineages in mouse embryonic stem cells expressing exogenous EGAM1 homeoprotein.

Embryonic stem (ES) cells have been considered as a valuable renewable source of materials in regenerative medicine. Recently, we identified the homeoprotein EGAM1 both in preimplantation mouse embryos and mouse ES cells. Expression of the Egam1 transcript and its encoded protein was detectable in differentiating mouse ES cells, while it was almost undetectable in undifferentiated cells. In the present study, in order to clarify the effect of forced expression of EGAM1 on the differentiation of mouse ES cells in vitro, transfectants expressing exogenous EGAM1 were generated. Egam1 transfectants promoted differentiation into cell types expressing Gata6, Gata4, Afp, or Plat, genes associated with emergence of the extra-embryonic endoderm lineages. On the other hand, Egam1 transfectants inhibited the expression of specific genes for the embryonic lineages, including Fgf5 (epiblast) and T (mesoderm), in addition to Cdx2, a specific gene for the extra-embryonic trophectoderm lineages. Changes in the percentage of cells recognizing by antibodies against specific marker proteins closely correlated with the expression patterns of their transcripts. Taken together, the results obtained in this study suggested that mouse ES cells expressing exogenous EGAM1 preferentially differentiate into extra-embryonic primitive endoderm lineages, rather than embryonic lineages or extra-embryonic trophectoderm lineages.

Common nucleotide sequence of structural gene encoding fibroblast growth factor 4 in eight cattle derived from three breeds.

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for the proper development of bovine embryos. However, the complete nucleotide sequences of the structural genes encoding FGF4 in identified breeds are still unknown. In the present study, direct sequencing of PCR products derived from genomic DNA samples obtained from three Japanese Black, two Japanese Shorthorn and three Holstein cattle, revealed that the nucleotide sequences of the structural gene encoding FGF4 matched completely among these eight cattle. On the other hand, differences in the nucleotide sequences, leading to substitutions, insertions or deletions of amino acid residues were detected when compared with the already reported sequence from unidentified breeds. We cannot rule out a possibility that the structural gene elucidated in the present study is widely distributed in cattle. To the best of our knowledge, this is the first determination of the complete nucleotide sequence of the structural gene encoding bovine FGF4 in identified breeds.

Effect of ectopic expression of homeoprotein EGAM1C on the cell morphology, growth, and differentiation in a mouse embryonic stem cell line, MG1.19 cells.

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, while Egam1c transfectants formed compacted colonies with increased E-CADHERIN expression. In Egam1c transfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genes Fgf5 (epiblast), T (mesoderm), Gata6 (primitive endoderm), and Cdx2 (trophectoderm) in -LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, including Tpbpa (spongiotrophoblast) and Plat (parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.

Production of mouse fibroblast growth factor 4 in E. coli and its application for isolation and maintenance of mouse trophoblast stem cells in vitro.

Fibroblast growth factor 4 (FGF4) promotes isolation of trophoblast stem (TS) cells from mouse blastocysts and maintenance of TS cells in an undifferentiated state in vitro. To date, commercially available, bacterially expressed human FGF4 (RhFGF4) has been used generally for this purpose. In this study, HismFGF4, a 6x histidine-tagged mouse FGF4, was produced in E. coli and purified using heparin column chromatography. We demonstrated that HismFGF4 (25 ng/ml) more efficiently generates mouse TS cells from a single blastocyst than RhFGF4 (25 ng/ml) and that TS cells isolated and maintained with HismFGF4 retained their ability to differentiate into the trophoblast cell lineage in vitro. In addition, TS cells cultured with HismFGF4 (25 ng/ml) were maintained in an undifferentiated state better than with RhFGF4 (25 ng/ml). To the best of our knowledge, this is the first application of a mouse FGF4 derivative for isolation and maintenance of mouse TS cells.

Identification of a 2-cell stage specific inhibitor of the cleavage of preimplantation mouse embryos synthesized by rat hepatoma cells as 5'-deoxy-5'-methylthioadenosine.

Rat hepatoma Reuber H-35 cells produce a unique compound designated as Fr.B-25, a 2-cell stage-specific inhibitor of the cleavage of preimplantation mouse embryos cultured in vitro. Here, we identified Fr.B-25 as a purine nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), by mass spectroscopic analysis. All of the biological activities examined of authentic MTA on the development of mouse zygotes were indistinguishable from those of Fr.B-25. The mechanism of MTA action in the development of preimplantation mouse embryos was probably different from those of hypoxanthine and adenosine, which are well-characterized purine nucleosides that act as inhibitors of the cleavage of mouse 2-cell embryos. From the shared molecular and biological properties of Fr.B-25 and MTA, we concluded that Fr.B-25 is MTA. To the best of our knowledge, this is the first delineation of the effect of MTA on the development of preimplantation mammalian embryos cultured in vitro.

Relationships between homeoprotein EGAM1C and the expression of the placental prolactin gene family in mouse placentae and trophoblast stem cells.

The mouse Crxos gene encodes three structurally related homeoproteins, EGAM1, EGAM1N, and EGAM1C, as transcription and splicing variants. Recently, we identified the functions of EGAM1 and EGAM1N in the regulation of differentiation in mouse embryonic stem cells. However, the function of EGAM1C remains unknown. To explore the additional roles of these proteins, the ontogenic expression of the respective mRNAs in post implantation mouse embryos and extraembryonic tissues, particularly from embryonic day (E) 10.5 to E18.5, was analyzed. The expression of Egam1n mRNA was specifically detected in embryos throughout this period, whereas that of Egam1 was undetectable in any of the tissues examined. However, in the placenta, Egam1c mRNA and its encoded protein were detected after E16.5, and these expression levels increased by E18.5 immediately before partum. Quantitative RT-PCR and in situ hybridization analyses in placentae revealed that the spatial and temporal expression patterns of the Egam1c mRNA were related to some extent with those of Prl3a1 and Prl5a1 and partially overlapped that of Prl3b1, which are members of the placental prolactin (PRL) gene family. When EGAM1C was overexpressed moderately in mouse trophoblast stem cells as a model for undifferentiated and differentiating placental cell types, the expression levels of endogenous Prl3b1 and Prl5a1 were enhanced under both undifferentiated and differentiating culture conditions. These results indicated that EGAM1C may play a role in the expression of members of the placental PRL gene family, such as Prl3b1 and Prl5a1.

Cloning of complementary DNAs encoding structurally related homeoproteins from preimplantation mouse embryos: their involvement in the differentiation of embryonic stem cells.

During the preimplantation development of mouse embryos between the 4-cell to 8-cell stage and the morula stage, when the first irreversible segregation of cell fates proceeds into the pluripotent inner cell mass (progenitor cells to form the fetus) and the trophectoderm (to form the placenta) of blastocysts, pluripotency-maintaining and differentiation-inducing genes are expressed to coordinately regulate cell fates. Three structurally related cDNAs (Crxos1, Crxos1 sv2, and Crxos1 tv3) that exhibited concomitant elevated expression during this critical period were identified by subtractive cDNA cloning. CRXOS1 contains two homeodomains, while CRXOS1 sv2 and CRXOS1 tv3 each contain one of the homeodomains included in CRXOS1. Crxos1, Crxos1 sv2, and Crxos1 tv3 were expressed differentially during in vitro embryonic stem (ES) cell differentiation. Even under differentiation-inducing conditions, forced expression of Crxos1 sv2 inhibited the differentiation of ES cells. In contrast, under conditions that promote self-renewal of ES cells, forced expression of Crxos1 induced differentiation. Forced expression of Crxos1 resulted in induction of Gata4 but in repression of T, probably indicating that Crxos1 promotes the differentiation of ES cells into primitive endoderm, while inhibiting differentiation into mesoderm. On the other hand, no apparent effects of forced expression of Crxos1 tv3 were observed. Taken together, it was concluded that these transcripts encoding homeoproteins are capable of regulating the maintenance and/or differentiation of mouse ES cells and likely regulate that of preimplantation embryos.

Effect of medium conditioned with rat hepatoma BRL cells on '2-cell block' of random-bred mouse embryos cultured in vitro.

The phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the beta-A or beta-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the beta-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria.

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the beta-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding beta-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes gamma-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited beta-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important beta-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

Characterization of the upper pathway genes for fluorene metabolism in Terrabacter sp. strain DBF63.

Genes involved in the degradation of fluorene to phthalate were characterized in the fluorene degrader Terrabacter sp. strain DBF63. The initial attack on both fluorene and 9-fluorenone was catalyzed by DbfA to yield 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone, respectively. The FlnB protein exhibited activities against both 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone to produce 9-fluorenone and 2'-carboxy-2,3-dihydroxybiphenyl, respectively. FlnD is a heteromeric protein encoded by flnD1 and ORF16, being a member of the class III two-subunit extradiol dioxygenase. FlnE was identified as a serine hydrolase for the meta-cleavage products that yield phthalate.

The rpoZ gene, encoding the RNA polymerase omega subunit, is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis.

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.