Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve

• DOI: 10.3389/fmicb.2021.636822
• Publication Date: 2021-03-23T06:54:37Z
• Authors: Emily C. Hoedt; Francesca Bottacini; Nora Cash; Roger S. Bongers; Kees van Limpt; Kaouther Ben Amor; Jan Knol; John MacSharry; Douwe van Sinderen
• Keywords: Bifidobacterium breve; Restriction site; Insertional mutagenesis; Shuttle vector

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due their extensive and variable repertoire Restriction-Modification (R-M) systems. Non-replicating plasmids currently employed achieve insertional mutagenesis in . One limitations using such insertion vectors is presence within sequence various restriction sites, making them sensitive activity endogenous endonucleases encoded by target strain. For this reason, have been developed with aim methylating protecting vector a methylase-positive Escherichia coli strain, some cases containing cloned bifidobacterial methylase. Here, we present approach based on modified synthetically produced version suicide pORI28 (named pFREM28), where all known sites targeted breve R-M systems were removed base substitution (thus preserving codon usage). After validating integrity erythromycin marker, was successfully an α-galactosidase gene responsible for raffinose metabolism, alcohol dehydrogenase mannitol utilization encoding priming glycosyltransferase exopolysaccharides (EPS) production B. The advantage reduction amount time, effort resources required generate site-directed mutants similar may be other ( bifido)bacterial species.