A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum

Abstract

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.

The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue

“…Nevertheless, such different adhesive roles played by the bifidobacterial SD fimbriome might be further influenced by the SD fimbriome or the extracellular structures produced by other members of the gut microbiota. Nevertheless, since many bifidobacteria are practically genetically inaccessible, with just a few exceptions (55,56), we are currently not in a position to test our predictions under in vivo circumstances. Future gene inactivation PEL experiments, when such procedures will become available, will therefore be important to confirm our in silico results.…”

The <i>pyrE</i> Gene as a Bidirectional Selection Marker in <i>Bifidobacterium Longum</i> 105-A

“…However, only a few genetic investigations have been reported for bifidobacteria, because sufficient gene manipulation techniques had not yet been developed for this genus. Recently, some useful gene manipulation techniques have been developed [4,5,6,7]. …”

Tough nuts to crack

“…In fact, in a later publication the authors admitted that the process was indeed very time consuming since it took more than one year to obtain a single clone of the mutant. 9 Accordingly, there are no further reports on mutants generated with this system in B. longum NCC2705 or other bifidobacteria.…”

“…38 Deletion of pyrE, which encodes orotatephosphoribosyltransferase and is crucial for pyrimidine metabolism, was confirmed by resistance to 5-fluoroorotic acid and auxotrophy for uracil of the mutant. The authors further validated their system by re-creating a deletion mutant in the bl0033 gene of B. longum NCC2705, 9 which was obtained earlier by the same group using a classical yet very time-consuming suicide vector strategy. 7 of apuB in B. breve UCC2003 encoding an extracellular type II amylopullulanase.…”

“…More recently, pKO403, a temperature-sensitive derivative of the E. coli/Bifidobacterium shuttle vector pKKT427, was successfully applied for the generation of two mutants in B. longum NCC27755. 9 The pKKT427 vector is based on the pTB6 replicon, which was shown to stably replicate in strains of B. longum, B. breve and B. animalis (reviewed in ref. 5).…”