A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains

Abstract

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3' end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.

Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export

“…Introduction of plasmids into B. amyloliquefaciens strains was carried out by E40 phage transduction or electrotransformation [22].…”

“…To construct plasmids that would deliver genetic modiWcations of interest into the chromosome of the naturally nontransformable B. amyloliquefaciens AJ1991, the thermosensitive rolling-circle replication pNZT1 plasmid, based on the pG + host replicon, was used as a vector [13,22]. To deliver the pbuE T!C mutation, plasmid pNZT1-pbuEm was constructed as follows.…”

“…The resulting fragment was BcuI-PstI-digested and cloned into BcuI-PstI -digested pNZT1, resulting in pNZT1-purH. Construction of marker-free genetically modiWed strains Genetic modiWcations were introduced into the chromosome of B. amyloliquefaciens using the previously described two-step replacement recombination procedure [22]. In the Wrst step, a Bacillus strain bearing the thermosensitive rolling-circle replication delivery plasmid containing the gene replacement construct was cultivated with aeration in LB at 37°C (a nonpermissive temperature for plasmid replication) to initiate integration of the entire plasmid into the chromosome.…”

“…The obtained fragment was digested with HindIII-XhoI and cloned into HindIII-XhoI-digested pNZT1-Ppur, yielding the pNZT1-Ppur-nepI plasmid. pNZT1-Ppur was constructed by cloning the 270-bp PstI-HindIII DNA fragment corresponding to the upstream region of the B. amyloliquefaciens pur operon between the PstI and HindIII sites of pNZT1 [22]. To incorporate the Ppur-nepI fusion into the chromosome of AJ1991, the aprE gene encoding serine alkaline protease was chosen as the target gene.…”

“…S. Sheremet · S. V. Gronskiy · R. A. Akhmadyshin · A. E. Novikova · V. A. Livshits · R. S. Shakulov · N. P. Zakataeva (&) Ajinomoto-Genetika Research Institute, 1-St Dorozhny Proezd, b.1-1, 117545 Moscow, Russia e-mail: natalia_zakataeva@agri.ru feature complicates genetic manipulation of these microorganisms and makes methods for obtaining recombinant strains relatively time and labor intensive. Recently, a routine method has been developed to introduce marker-free deletions, insertions or point mutations into chromosomes of Bacillus strains, including those that are naturally nontransformable [22]. The method is based on a combination of eVective plasmid transfer and the replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid as the delivery vector.…”