Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

Abstract

Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCEThe nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia. Botulism, caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum, is a rare but severe paralytic illness in humans and animals. Botulinum toxins are 150-kDa proteins with zinc endopeptidase activity, consisting of two subunits, a 100-kDa heavy chain and a 50-kDa light chain. The heavy chain is responsible for binding and translocation of the light chain into the cytosol of neuronal cells, whereas the light chain cleaves SNARE proteins that are involved in the docking of acetylcholine-containing vesicles and fusion to the presynaptic membrane. When the SNARE proteins are cleaved, neurotransmitter release is inhibited, leading to the paralysis of the corresponding muscle (1, 2).C. botulinum is a strictly anaerobic bacterium that thrives in decaying organic matter in soils and sediments of ponds, lakes, and oceans. It also forms dormant endospores that are highly resilient to hostile conditions and therefore can be found widespread in the environment. These spores may contaminate foods via raw materials or other sources, possibly leading to food-borne botuli...

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

“…This work was conducted in an atoxigenic (Δ bont :: ermB ) and uracil auxotrophic (Δ pyrE ) mutant of group II C. botulinum strain NCTC 11219 type E, further in this manuscript shortly named Δ bont Δ pyr or parent strain 34 . This strain was used for biosafety reasons, and because of the possibility to use pyrE in trans as a negative selection marker for replacement of the gerBAC locus.…”

“…Spore suspensions were handled in open air, and transferred to the workstation only for experiments involving outgrowth. Spore suspensions were prepared using a two-phase sporulation medium and stored in 0.85% NaCl at 4 °C as described previously 34 . E. coli strains were grown in lysogeny broth (LB; 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) or on LB agar (LB + 15 g/l agar) at 37 °C.…”

“…Plasmid pMTL84151∆ gerBAC was introduced into NCTC 11219 Δ bont Δ pyr by conjugation as described previously 34 , using selection on RCM agar with Tm and Cy. Purified transconjugants were resistant to Sp and sensitive to FOA, confirming expression of the plasmid aad9 resistance marker and pyrE gene, respectively.…”

“…In the present work, we deleted the entire gerBAC locus encoding GerX3b to analyse its role in germination by different nutrient and non-nutrient germinants. This was done in a mutant of gIICb strain NCTC 11219 from which we previously deleted the bont/E toxin gene 34 . Gene deletion is still a major technical challenge in gIICb, and the present work is only the second (after the deletion of bont/ E) in which this is successfully accomplished.…”

Exploring the Ambiguous Status of Coagulase-Negative Staphylococci in the Biosafety of Fermented Meats: The Case of Antibacterial Activity Versus Biogenic Amine Formation

“…Eighteen antibacterial staphylococcal strains were tested for antagonistic activity against a total of 33 C. botulinum strains, (both group I and group II; Supplementary Table S3), using a spot-on-lawn assay [47,48]. All manipulations of C. botulinum were carried out in an anaerobic workstation [Whitley DG250 (Don Whitley Scientific, Bingley, UK); 80% N 2 , 10% CO 2 , and 10% H 2 ] at 30 • C. All media/solutions were deoxygenated prior to use by anaerobic incubation overnight.…”