Generation of Targeted Deletions in the Genome of Rhodothermus marinus

Abstract

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp ؉ selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade ؉ selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (⌬trpB ⌬purA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (⌬trpB ⌬purA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5 and 3 homologous sequences, respectively. The resulting Trp ؉ transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.

Characterization of carotenoids in Rhodothermus marinus

“…The DPPH assay showed 3‐ to 3.7‐fold decrease in antioxidant capacity of R. marinus SB‐71 extract in comparison to the carotenoid‐producing strains, while the TEAC assay showed a 1.7 to 1.9‐fold decrease. The difference between R. marinus strain PRI 493 and SB‐71 is the knock‐out of several carotenoid pathway genes (Bjornsdottir et al., ) and the mutant could therefore be used as a negative control during the antioxidant capacity assays, meaning that any difference between these two strains in antioxidant capacity is due to the presence or absence of the carotenoid biosynthetic pathway (Bjornsdottir et al., ). To the best of our knowledge, antioxidant capacity has neither been studied for the carotenoids produced by R. marinus nor for the structurally similar carotenoids of S. ruber .…”

“…A few genes of the pathway have previously been determined through knock‐out trials in R. marinus strain SB‐71. Strain SB‐71 ( ΔtrpBΔpurAcrtBI’::trpB ) has two carotenoid biosynthetic genes ( crtB and crtI) knocked out, which normally catalyze two units of geranylgeranyl pyrophosphate to phytoene and phytoene to lycopene, respectively (Bjornsdottir et al., ). These mutations resulted in white colonies in contrast to its natural red color due to the absence of carotenoids.…”

“…This gives way for further opportunities, including genetic engineering for the production of carotenoid derivatives. (Bjornsdottir, Fridjonsson, Hreggvidsson, & Eggertsson, ) The possibility of production of natural or altered carotenoids highlights the importance of the understanding of the carotenoid biosynthetic pathway. A few genes of the pathway have previously been determined through knock‐out trials in R. marinus strain SB‐71.…”

Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

“…Because rpsL-based gene deletion only works in a streptomycin-resistant mutant strain, sacB was established as the preferred counterselectable marker for construction of in-frame deletion mutants. Previous attempts to use sacB as a counterselectable marker in members of the phylum Bacteroidetes failed, perhaps because of a lack of expression of the sacB gene (18,19). We eliminated this problem by introducing a Bacteroidetes promoter that is known to function in many members of the phylum.…”

“…In some other Gram-negative bacteria, sacB, which confers sensitivity to sucrose, has been used as a counterselectable marker to allow isolation of gene deletion mutants from wild-type cells (15)(16)(17). Previous attempts to adapt this approach to F. johnsoniae or to other members of the phylum Bacteroidetes were not successful (18,19). …”