Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

Abstract

Thermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase ␣-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog pchlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on ␤-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well. IMPORTANCEThermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into the T. thermophilus genome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S

“…Mutating the conserved alanine corresponding to A294 in the PheS of Escherichia coli to glycine was previously shown to generate an effective counterselection system in E. coli, Enterococcus faecalis, Streptococcus mutans, and Thermus thermophilus (12)(13)(14)(15). Using Clustal Omega, we aligned the amino acid sequences of PheS from E. coli to those of Listeria monocytogenes, Listeria innocua, and Bacillus subtilis and found the conserved alanine in all species (Fig.…”

Curing the Megaplasmid pTT27 from Thermus thermophilus HB27 and Maintaining Exogenous Plasmids in the Plasmid-Free Strain

“…This plasmid-free white strain with natural competence might have great potential as a host strain for DNA engineering in T. thermophilus. The absence of a plasmid in the host cell is attractive for plasmid transformation, and white colonies are suitable for color-based selection, such as yellow caused by carotenoid production (13) or blue caused by X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) and ␤-galactosidase (15). In this aspect, the PFW I-RNR strain, in which the megaplasmid-originated class I RNR genes were carried in the chromosome, would be better for use as the host cells, because it improves growth and stably maintains exogenous plasmids (Fig.…”

“…The bacteria themselves have also been extensively investigated as model organisms for systems biology including structural genomics (1, 2), and their genomic DNAs, with GϩC contents as high as about 70%, are an interesting target for synthetic biology (3). Thermus-related studies have been encouraged by the existence of an established genetic engineering system based on natural competence (4-6), with thermostabilized drug resistance markers (7-10) and other thermostable genetic tools (1,(11)(12)(13)(14)(15)(16).Complete genomic sequences of T. scotoductus SA-01, T. oshimai JL-2, Thermus sp. strain CCB_US3_UF1, T. aquaticus Y51MC23, T. thermophilus strains HB8, HB27, SG0.5JP17-16, and JL-18, and others are available online.…”

Phenylalanyl-tRNA synthetase

“…This conditionally lethal mutant has been used as a counter selection marker for introducing genomic point mutations or deletions in T. thermophilus. 136 To date, the mechanism of aaRS mutation …”