Kano Kasuga

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites.

Abstract. Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.

Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features.

ABSTRACT Two kinds of bacteria having different-structured angular dioxygenases—a dibenzofuran (DF)-utilizing bacterium,Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10—were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.

Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1

Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO2) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon5 (Tn5) mutagenesis had a Tn5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in Terrabacter sp. strain DBF63.

Abstract. Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene (bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53–62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.

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.

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.

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.

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.

Factors Affecting the Expression of Differentiation Marker Genes for the Primitive Endoderm Lineage in a Mouse Extra-Embryonic Endoderm Stem Cell Line, XEN26 Cells

ABSTRACT The extra-embryonic endoderm lineages, visceral endoderm (VE) and parietal endoderm (PE), comprise part of the yolk sac and support embryonic development. Mouse extra-embryonic endoderm stem (XEN) cells were established from primitive endoderm (PrE), the founder cells of VE and PE, from preimplantation blastocysts. The differentiation of XEN cells is usually induced in adherent conditions without medium conditioned by mouse embryonic fibroblasts (MEFCM), which maintains self-renewal of the cells. In this study, we analyzed the effects of suspension culture and retinoic acid (RA) treatment on the differentiation of XEN26 cells, a mouse XEN cell line, toward PrE derivatives. The suspension culture without both MEFCM and RA promoted the expression of α-fetoprotein (Afp) mRNA, a specific marker for VE, at 3- to 4-fold higher levels (P < 0.05) than those in adherent culture. In these conditions, the cellular content of hepatocyte nuclear factor-4α (HNF4α) protein, a crucial transcription factor involving Afp expression, increased, while that of HNF1α was relatively constant. On the other hand, RA treatment induced upregulation of the mRNA for tissue plasminogen activator (Plat), a specific marker for PE, in both adherent and suspension culture conditions. From these results, our new attempts, including the application of RA treatment and suspension culture, for the induction of differentiation in XEN26 cells led to significant upregulation of Plat and Afp, respectively.

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.