The body wall collagen of an edible sea cucumber, Stichopus japonicus, was studied with respect to its chemical composition and subunit structure. About 70% of the total body wall protein was accounted for by highly insoluble collagen fibers. The disaggregation with b-mercaptoethanol, 0.1 M NaOH treatment, and limited pepsin digestion of these collagen fibers resulted in complete solubilization. The solubilized collagen was isolated and characterized; it had 2 distinct subunits, a1 and a2, which formed (a1) 2 a2 heterotrimers and was rich in glutamic acid when compared with other fibrillar collagens. The unique textural properties of cooked sea cucumber seem to be due to thermal denaturation of the insoluble collagen fibers.

A nonylphenol-assimilating bacterium isolated at a sewage-treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of the 16S rDNA sequence, the GMC content of the DNA (63 mol %) and the isoprenoid quinone composition, as well as the presence of sphingoglycolipid and the whole-cell fatty acid profile, revealed that the isolate is a member of the genus Sphingomonas. However, the sequence similarity of the 16S rDNA with that of known Sphingomonas spp. was found to be at most 96 %, implying that the isolate is distinctive. Furthermore, the results of DNA-DNA hybridization experiments and its physiological characteristics clearly indicated that the isolate represents a new Sphingomonas species, for which the name Sphingomonas cloacae is proposed ; strain S-3 T (l JCM 10874 T l IAM 14885 T ) is the type strain.Keywords : Sphingomonas cloacae sp. nov., biodegradation, endocrine disrupter chemical, nonylphenol INTRODUCTIONNonylphenol (NP) is known as an important intermediate in the production of many kinds of commercial and industrial materials. Above all, NP is used in the production of nonylphenol polyethoxylate, a nonionic surfactant with many industrial applications. However, NP is also known as a ubiquitous pollutant in urban aquatic environments, with concentrations in the p.p.b. (µg l −" ) order, and is found in both sediment and surface water (Giger et al., 1984 ;Ahel & Giger, 1985 ;Brunner et al., 1988 ;Marcomini et al., 1990 ;Rudel et al., 1996 ;Isobe & Takada, 1998 ;Kojima & Watanabe, 1998). There is growing evidence that NP has a certain oestrogenic activity (Granmo et al., 1989 ;Ekelund et al., 1990 ;Soto et al., 1991 ;Ahel et al., 1993 ; Jobling & Sumpter, 1993 ;Gray & Metcalfe, 1997 ;Ren et al., 1997 ;Shurin & Dodson, 1997 ;Ashfield et al., 1998 ;Coldham et al., 1998 ;Sonnenschein & Soto, 1998 Abbreviation : NP, nonylphenol.The GenBank/DDBJ accession number for the 16S rDNA sequence of strain S-3 T is AB040739. et al., 2000) and have isolated a NPdegrading bacterium (K. Fujii and others, unpublished data), which has been designated strain S-3 T . This strain degraded 1000 p.p.m. NP, i.e. a 10 000-1 000 000-fold higher concentration than found in urban environments, almost completely within 10 d. Analysis of 16S rDNA partial sequences strongly suggested that the bacterium is a novel species of the genus Sphingomonas. Tokyo (FujiiTo precisely identify and phylogenetically place strain S-3 T , phenotypic characterization, analysis of isoprenoid quinone composition, fatty acid composition, polar lipid pattern and DNA GjC content, 16S rDNA sequencing and DNA-DNA hybridization were carried out. On the basis of our results we propose that S-3 T should be placed in a new species of the genus Sphingomonas, Sphingomonas cloacae. METHODS Chemicals* IFO, Institute for Fermentation, Osaka, Japan ; JCM, Japan Collection of Microorganisms, Saitama, Japan ; ATTC, American Type Culture Collection, Manassas, VA, USA ; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen...

All five species in the heterotrophic micro-algal genus Prototheca and their relatives were compared for the extent of nucleotide divergence in the nuclear small-subunit (SSU) and in the 5' end of large-subunit (LSU) ribosomal RNA genes (rDNAs). Phylogenetic analysis based on combined SSU and LSU rDNA sequence alignment was implemented with the neighbor-joining, the maximum-parsimony, and the maximum-likelihood methods. The relationships among the species of Prototheca based on this data set were largely concordant with those inferred from SSU or LSU rDNA sequences alone. The obtained phylogenetic trees indicated that P. stagnora and P. ulmea should be regarded as different species and that both of the species as well as P. moriformis were placed in a cluster represented by P. zopfii, whereas P. wickerhamii was not directly grouped together with the other members of Prototheca and was more closely related to the autotrophic alga Auxenochlorella protothecoides. Therefore, the genus Prototheca is paraphyletic in its present circumscription; and these conclusions lead us to propose the transfer of P. wickerhamii to Auxenochlorella or to a new genus. On the basis of nucleotide sequence similarities, unlike SSU rDNA, the LSU rDNA region examined in this study appeared to be variable in recognizing a heterogeneity within a single species P. zopfii, which had been shown earlier in a chemotaxonomic study.

SUMMARY: Matrix metalloproteinases (MMP) are widely distributed in vertebrate tissues. One of the well‐characterized gelatinases from higher vertebrates is MMP‐2, named gelatinase A or 72 kDa type IV collagenase, which is active for the cleavage of denatured collagens, type IV collagen, type V collagen, and other matrix proteins. To investigate the primary structure and properties of MMP‐2 from teleost as lower vertebrates, a cDNA library was prepared from mRNA of rainbow trout fibroblast and screened. Using polymerase chain reaction and degenerate oligonucleotide primers, which are specific for two highly conserved sequences found in MMP of higher vertebrates, a resultant cDNA fragment was used as a probe. A cDNA clone 3.0 kb long was isolated and found to contain an open‐reading frame coding for a polypeptide of 655 amino acids. The rainbow trout polypeptide was 73% identical, at the level of amino acid sequence, to human proMMP‐2 with the greatest degree of similarity occurring in the propeptide and catalytic domains and was denoted as rainbow trout proMMP‐2. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and human type V collagen, providing support to the hypothesis that the cDNA codes for the authentic rainbow trout proMMP‐2. In contrast to human proMMP‐2, rainbow trout proMMP‐2 was not activated by 4‐amino‐phenylmercuric acetate. This is the first report of cDNA for fish proMMP to our knowledge.

Nonylphenol (NP) is an important intermediate in the production of various commercial and industrial materials, but is also known as a ubiquitous pollutant in urban aquatic environments. We recently studied the NP-degrading activities of microflora in several aquatic environments, and found a notable degrading activity for wastewater from a sewage treatment plant in Tokyo. This result led us to isolate NP-degrading microbes and identify biodegradation products. Using conventional plate culture techniques and molecular biological methods, Pseudomonas and Sphingomonas species, which are known for their degradation activities of many aromatic compounds, have been isolated. But it has also been found that Sphingomonas sp. (S-strain) is necessary and sufficient for the degradation of NP. Although the role of Pseudomonas sp. (P-strain) remains unclear, P-strain seems to provide some co-nutrients for the growth of S-strain. The degradation products were analyzed by GC/MS and NMR. More than 95% of NP was degraded within 10 days and aromatic compounds other than NP were not found, suggesting that the phenolic part of NP was completely degraded. We also examined the potential of S-strain for bioremedial applications. S-strain cells immobilized on chitosan or alginate beads retain their NP-degrading activity in flask-scale experiments. Furthermore, the chitosan-bound cells in a lab-scale bioreactor have been found to be persistent for repeated use, suggesting that S-strain is applicable to the treatment of NP-contaminated wastewater.