Analysis and characterization of eight estradiol inducible genes and a strong promoter from the steroid degrading marine bacterial strain S19-1

Abstract

Buttiauxella strain S19-1 is a new marine bacterium, isolated from the Baltic Sea, which can degrade steroids. In this report, a meta-genomic approach was used to isolate estradiol inducible genes from S19-1. SalI-fragments from the chromosomal DNA of S19-1 were ligated into plasmid pKEGFP2 bearing an EGFP gene as the reporter system. All resulting plasmids harboring SalI-fragments were transformed into Escherichia coli HB101 to measure the relative fluorescent units (RFU). E. coli cells showing higher RFU after estradiol induction than those without estradiol induction, were selected and the respective plasmids were sequenced. Sequences of 8 positive plasmids were analyzed and aligned by BLAST. Among the predicted genes we found similarities to the major facilitator superfamily, glycerol dehydratase activator, formate acetyltransferase activating enzyme, histidinol-phosphate/aromatic aminotransferase, ABC-transporter, transcriptional regulator nadR, lipoate-protein ligase A, and alcohol phosphatidyl-transferase. Interestingly, one of the E. coli cell clones (containing plasmid p302) showed up in green color by normal light microscopy, which indicated that a strong promoter was present in this plasmid. Sequencing and deletion-mutagenesis revealed that the putative promoter comprises a 108bp DNA fragment within p302, from which the putative −10 and −35 regions are TTTGAT and TTGGTT, respectively. The promoter might be used to construct S19-1 mutants in which steroid degradation occurs at high levels.