Abstract
Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.
Highlights
Bifidobacteria are common gut commensals that have been reported to elicit a number of beneficial effects on their host (Tojo et al, 2014; O’Callaghan and van Sinderen, 2016; Wong et al, 2019)
One of the major limitations in using commonly available insertion vectors for targeted mutagenesis is the presence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain (O’Connell Motherway et al, 2009; Bottacini et al, 2018b)
This methylation can be achieved in two ways: (i) the plasmid construct is introduced into a methylase positive Escherichia coli strain which may express a cloned methylase from an active R-M system present in the bifidobacterial target strain; (ii) the less frequently employed chemical methylation
Summary
Bifidobacteria are common gut commensals that have been reported to elicit a number of beneficial effects on their host (Tojo et al, 2014; O’Callaghan and van Sinderen, 2016; Wong et al, 2019). In order to facilitate the introduction of pORI19-tet into a target strain at sufficiently high frequency to allow gene disruption by homologous recombination an additional step of methylation of the vector is required This methylation can be achieved in two ways: (i) the plasmid construct is introduced into a methylase positive Escherichia coli strain (e.g., the DAM+ E. coli EC101) which may express a cloned methylase from an active R-M system present in the bifidobacterial target strain; (ii) the less frequently employed chemical methylation. This multi-step approach, despite being successfully applied previously, presents some practical hurdles and limitations: first of all the intermediate step of methylase cloning and methylation of the vector in E. coli is quite laborious and time consuming, secondly the system may not always be applicable in cases where target strains contain multiple active R-M systems or in cases where there is no information available on the R-M systems of the target strain
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