Identification of a new steroid degrading bacterial strain H5 from the Baltic Sea and isolation of two estradiol inducible genes

Abstract

a b s t r a c tThe presence of steroid hormones in the aquatic environment is potentially threatening the population dynamics of all kinds of sea animals and public health. Environmental estrogens in water have been reported to be associated with abnormal sexual development and abnormal feminizing responses in some animals. New approaches for the bioremediation of steroid hormones from the environment are therefore urgently sought. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. In the present investigation, 16S rRNA analysis showed that marine strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. To enable identification of steroid inducible genes from bacterial strain H5, a library was constructed of H5 chromosomal DNA fragments cloned into a fluorescent reporter (pKEGFP-2). A reporter plasmid pK3␣-4.6-EGFP3 containing the estrogen-inducible gene 3␣-hydroxysteroid dehydrogenase/carbonyl reductase (3␣-HSD/CR) from Comamonas testosteroni (C. testosteroni) was created as a positive control. Steroid induction could be detected by a microplate fluorescence reader, when the plasmids were transformed into Escherichia coli (E. coli) HB101 cells. With our meta-genomic pKEGFP-2 approach, we identified two estradiolinducible genes from marine strain H5, which are obviously involved in steroid degradation. Sequencing of the pKEGFP-2 inserts and data base research at NCBI revealed that one gene corresponds to 3-ketosteroid-delta-1-dehydrogenase from several Mycobacterium strains, while the other showed high similarity to carboxylesterase in Sebadella termitidis and Brachyspira murdochii. Both 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase are one of the first enzymes in steroid degradation. In addition, we identified a strain H5 specific DNA sequence of 480 bp which allows sensitive PCR detection and quantification of strain H5 bacteria in "unknown" seawater samples. Currently, the exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged, which might be used for the bioremediation of steroid contaminations in seawater.Article from a special issue on steroids and microorganisms.

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5

“…1a). A 4.610 kb DNA fragment containing the 3␣-hydroxysteroid dehydrogenase/carbonyl reductase (3␣-HSD/CR) gene together with its regulatory region from the C. testosteroni chromosomal DNA was cloned into the plasmid pKEGFP-2 at its BamHI and SalI sites to yield plasmid pK3␣-4.6-EGFP3 [25]. Plasmids pKEGFP-H5-38S2 and pKEGFP-H5-24C2 harbouring the genes coding for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were constructed from pKEGFP-H5-38 and pKEGFP-H5-24 by SphI-Xbal and SalI-ClaI digestion, respectively (Fig.…”

“…Plasmids pKEGFP-H5-38S2 and pKEGFP-H5-24C2 harbouring the genes coding for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were constructed from pKEGFP-H5-38 and pKEGFP-H5-24 by SphI-Xbal and SalI-ClaI digestion, respectively (Fig. 1b) [25]. For overexpression and purification of the estradiol inducible enzymes 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase from the marine bacterium strain H5, E. coli strain BL21(DE3)plysS together with plasmid pET15b from Novagen was used.…”

“…In previous investigations, by using plasmid pKEGFP-2 (negative control) and plasmid pK3␣-4.6-EGFP3 (positive control) in an EGFP meta-genomic system, two estradiol inducible genes coding for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were identified [25]. For this purpose, the EGFP gene had been cloned downstream from the 3␣-HSD/CR gene to yield plasmid pK3␣-4.6-EGFP3.…”

“…Gram-staining and 16S rRNA analysis revealed that strain H5 is bacilliform in shape and belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria [25]. Moreover, by employing a new meta-genomic system with the gene coding for the short-chain dehydrogenase/reductase (SDR) Fig.…”

“…As shown in part b, Plasmids pKEGFP-H5-38S2 and pKEGFP-H5-24C2 harbouring the genes coding for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were obtained from pKEGFP-H5-38 and pKEGFP-H5-24 by SalI-ClaI and SphI-Xbal digestion, respectively. enzyme 3␣-hydroxysteroid dehydrogenase/carbonyl reductase (3␣-HSD/CR), we have successfully isolated two estradiol-inducible genes from the strain H5 genome [25]. These two genes code for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively.…”