Genetic tools for the investigation of Roseobacter clade bacteria

Abstract

Background: The Roseobacter clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools.

Results: Conjugation and electroporation methods for the efficient and stable genetic transformation of selected Roseobacter clade bacteria including Dinoroseobacter shibae, Oceanibulbus indolifex, Phaeobacter gallaeciensis, Phaeobacter inhibens, Roseobacter denitrificans and Roseobacter litoralis were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene dnr from D. shibae DFL12T.

Conclusion: A genetic toolbox for members of the Roseobacter clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria.

Figure 1

Flavin-based fluorescent protein as reporter gene. Fluorescence quantification of pRhokHi-2-FbFP-containing Roseobacter bacteria. Liquid cultures (MB, 48 h, 30°C, 200 rpm) were diluted in MB to an OD₅₇₈ of 0.7 and excited at 450 nm in a luminescence spectrometer LS 50 B from Perkin Elmer. The fluorescence emission was detected at 475 - 550 nm. Cultures of wildtype strains were used as a negative control. Results are expressed as mean values of three independent measurements.

Figure 2

Gene knockout strategy in D. shibae DFL12^T. (A) Schematic presentation of the dnr locus of D. shibae DLF12^T wildtype and the corresponding Δdnr-mutant. The deletion of Dshi_3189 (dnr) after homologous recombination into the D. shibae DFL12^T genome was confirmed by (B) PCR of D. shibae DFL12^T (line 1) and the Δdnr knockout mutants (line 2 and 3), using the primers oPT19 and oPT22 and by (C) growth of D. shibae DFL12^T and two Δdnr knockout mutants in MB supplemented with 25 mM nitrate under anaerobic conditions at 30°C and 100 rpm. Shown are the growth curves of D. shibae DFL12^T (-■-), D. shibae DFL12Δdnr1 (-□-) and D. shibae DFL12Δdnr2 (-Δ-).