Through Laboratory Domestication, Clostridium Botulinum Group II Strain Beluga Exhibits Genomic and Phenotypic Diversity

Abstract

The genetic and physiological alterations of organisms obtained through several in vitro passages lead
to laboratory domestication. Higher organisms and microorganisms have both been shown to exhibit
this phenomena. We examined several spore stocks of Clostridium botulinum Group II Beluga from our
collection in an attempt to comprehend the effects of laboratory domestication on the foodborne
pathogen and associated microbial food safety research, since that is a commonly used model strain
that has been used in labs for decades. Using phenotypic microarrays, an acquired nutritional
auxotrophy was verified as thymidine dependency. Parallel to this, whole-genome re-sequencing of all
stocks showed that the auxotrophic stocks had a mutation in thyA, which codes for thymidylate
synthase, which is necessary for the de-novo synthesis of dTMP from dUMP. By successfully
introducing an intact version of thyA into a thyA-deficient Beluga variant stock, thymidine prototrophy
was recovered, demonstrating that the thymidine auxotrophy was only caused by an SNP in thyA.
According to our data, this mutation has been present and sustained in laboratory stocks for almost 30
years. It is harmful when growth circumstances are inadequate in terms of nutrients and in a chemically
defined media. However, since obtaining the strain, the mutation has remained unknown, most likely as
a result of routinely using culture conditions that are best suited for growth performance. This study
highlights the necessity of closely observing model strains that are frequently utilized in lab settings at
the phenotypic and genomic levels. Compromised strains have the potential to produce inaccurate
predictions in applications such as food safety challenge testing, which could have detrimental effects.
We advise avoiding single-colony passaging and maintaining low passage numbers of laboratory
strains to reduce the chance of acquiring mutations. Furthermore, to rule out DNA mutations that could
compromise the integrity and repeatability of study data, pertinent strains should undergo routine WGS
analyses and physiological validation.