[Cloning and characterization of gene cluster for biosynthesis of ectoine and 5-hydroxyectoine from extreme halotolerant actinomycete strain Prauserella alba YIM 90005(T)].

ABSTRACT

OBJECTIVE: To study adaptive mechanism in hypersaline environments of extreme halotolerant filamentous actinomycetes. METHODS: Using HPLC we analyzed compatible solutes from extreme halotolerant filamentous actinomycete strain Prauserella alba YIM 90005T that was cultivated at different NaCI concentrations. RESULTS: Ectoine and 5-hydroxyectoine were two major compatible solutes for strain Prauserella alba YIM 90005T. Ectoine accumulated to the maximum content of 18.77 microg/mg dry cell weight after being inoculated in 10% NaCl (w/v). And 5-hydroxyectoine reached 22.98 microg/mg dry cell weight after being inoculated in 24% NaCl (w/v). The ectA (acyltransferase), ectB (aminotransferase), ectC (ectoine synthase) and ectD (ectoine hydroxylase) genes cluster encoding genes on ectoine and hydroxyectoine synthesis were further cloned by designing the degenerate primer and genome walking methods. The sequence analysis indicated that ectABCD was an operon. Furthermore, the expression of ectB and ectD inoculated at different salt concentrations was quantified by real-time PCR, and the results indicated that the expression of the gene cluster would be increasing as the salt concentration increased. CONCLUSION: 5-hydroxyectoine was the major compatible solute for osmotic regulation of strain Prauserella alba YIM 90005T to adapt high salt concentration.