Preliminary examinations for applying a carbazole-degrader, Pseudomonas sp. strain CA10, to dioxin-contaminated soil remediation

A method for bioremediation of chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) by a carbazole-utilizing bacterium, Pseudomonas sp. strain CA10, was developed. CA10 cells transferred to carbon- and nitrogen-free mineral medium supplemented with 1 mg carbazole (CAR)/ml grew rapidly during the first 2 days; and the cells at the end of this rapid growth period showed the highest 2,3-dichlorodibenzo-p-dioxin (2,3-Cl2DD)-degrading activity. The CA10 cells pregrown for 2 days efficiently degraded 2,3-Cl2DD in aqueous solution at either 1 ppm or 10 ppm. The effect of inoculum density on the efficiency of 2,3-Cl2DD degradation was investigated in a soil slurry microcosm [ratio of soil:water = 1:5 (w/v)]. The results showed that a single inoculation with CA10 cells at densities of 10(7) CFU/g soil and 10(9) CFU/g soil degraded 46% and 80% of 2,3-Cl2DD, respectively, during the 7-day incubation. The rate of degradation of each CDD congener, 2-ClDD, 2,3-Cl2DD, and 1,2,3-Cl3DD (1 ppm each) by strain CA10 in the soil slurry system was not significantly influenced by the coexistence of the other congeners. Using this soil slurry system, we tried an experimental bioremediation of the actual dioxin-contaminated soil, which contained mainly tetra- to octochlorinated dioxins. Although the degradation rate of total CDD and CDF congeners by a single inoculation with CA10 cells was 8.3% after a 7-day incubation, it was shown that strain CA10 had a potential to degrade tetra- to hepta-chlorinated congeners including the most toxic compound, 2,3,7,8-tetrachlorinated dibenzo-p-dioxin.     

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.     

Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor

Hemophores are secreted by several gram-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Yersinia pestis) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA(SM)), the P. fluorescens hemophore HasA(PF) has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA(PF) undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR(SM). Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR(SM). To localize the hemophore regions involved in binding to HasR, chimeric HasA(PF)-HasA(SM) proteins were tested for biological activity. We show that residues 153 to 180 of HasA(PF) are necessary for its interaction with the receptor.     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Coexistence of Anadromous and Resident Life History Styles of Pond Smelt, <Emphasis Type="Italic">Hypomesus nipponensis</Emphasis>, in Lake Ogawara,Japan, as determined by Analyses of Otolith Structure and Strontium:Calcium Ratios

Anadromous and resident forms of pond smelt, Hypomesus nipponensis, were found to occur in sympatry in Lake Ogawara, Japan. Profiles of Sr: Ca ratios from individuals could be grouped to two patterns (1) a resident pattern with low Sr: Ca ratios from core to edge and (2) an anadromous pattern with relatively low Sr: Ca ratios near the core with abrupt increases in ratios at a location approximately 0.3mm from the core. Spawners smaller than 60mm standard length (SL) were resident, between 60 to 80mm were mixed resident and anadromous, and larger than 80mm were anadromous. Anadromous individuals first migrated after 40 to 82 days from hatching (mean±sd, 59.1±13.5d) and 14.6 to 30.9mm SL (22.2±5.3mm). There was no difference in SL between resident and anadromous individuals during age at first migration, suggesting that size may not be the mechanism for divergence of alternative life history styles.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.