Changes in the genomes and methylomes of three Salmonella enterica serovars after immersion in black pepper for six years

Authors: Davies, Cary; JURKIW, THOMAS - Food And Drug Administration(FDA); HAENDIGES, JULIE - Food And Drug Administration(FDA); REED, ELIZABETH - Food And Drug Administration(FDA); ANDERSON, NATHAN - Food And Drug Administration(FDA); HOFFMAN, MARIA - Food And Drug Administration(FDA); ZHENG, JIE - Food And Drug Administration(FDA)

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2022
Publication Date: 9/9/2022
Citation: Davies, C.L., Jurkiw, T.J., Haendiges, J., Reed, E., Anderson, N., Hoffman, M., Zheng, J. 2022. Changes in the genomes and methylomes of three Salmonella enterica serovars after immersion in black pepper for six years. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2022.970135.
DOI: https://doi.org/10.3389/fmicb.2022.970135

Interpretive Summary: Low moisture foods (LMFs), or those with a low water content, include cereals, grains, oils, nuts, and spices, among others. Foodborne outbreaks associated with LMFs are increasing, and the foodborne bacteria Salmonella enterica is frequently implicated. S. enterica can survive for years in LMFs, but the mechanisms by which this pathogen tolerates desiccation, low nutrients, and other stressors are not well understood. Rates of DNA mutation may increase in response to stress, and while some resulting mutations may be disadvantageous, others may enhance fitness and survival. Here, we document DNA mutations in the genomes of three S. enterica serovars after storage in dried black pepper for six years. We also characterize patterns of methylated DNA across the genomes, the methylomes, and determine whether changes in methylation occur after immersion in black pepper. DNA methylation is involved in numerous cellular processes including virulence, DNA replication and repair, and gene expression, and thus may enhance survivability of S. enterica in the pepper matrix. In total, we identified nine single nucleotide mutations across the three serovars. We further cloned and characterized one mutation, a deletion in the gene mazG, which is known to play a role in bacterial adaptation to stress. Growth of the mazG mutant and the wild type (WT) strain were similar, but the WT strain outperformed the mutant under 16 conditions tested. In contrast, the mutant grew better than the WT in the presence of a small number of metabolites including the carbon sources methyl pyruvate and D-lactic acid methyl ester, suggesting that the mazG mutation may confer an advantage under some environmental conditions. In addition, we identified nine distinct methylated DNA motifs across the three serovars including five which were previously known, and four which are novel and serovar-specific. No changes in the number of each methylated motif were observed after immersion in pepper, except there was an increase in the number of BATGCAT motifs which were methylated. These findings, coupled with additional studies which further explore the functional consequences of the changes, will provide a basis for implementing appropriate interventions to eliminate the presence of S. enterica in LMFs and safeguard the food supply.

Technical Abstract: Low moisture foods (LMFs) have low water activity, aw, and have traditionally been recognized as safe for consumption, as most bacteria require higher water content to grow. However, outbreaks among LMF foods are increasing, and the microbial pathogen Salmonella enterica is implicated in the majority of these outbreaks. The mechanisms by which S. enterica survives in LMF environments are not well understood. Here, we examine changes in the genomes and methylomes of three serovars of Salmonella enterica subsp. enterica after immersion in the LMF black pepper for six years. A small number of single nucleotide polymorphisms were observed across the three surviving serovars including six substitutions (4 synonomous, two non-synonomous), one substitution in a non-coding region, and two insertion-deletions. No changes in gene presence-absence were observed. In total, nine distinct N6-methyladenine (m6A) methylated motifs across the three serovars were identified including five which were previously known, Gm6ATC, CAGm6AG, BATGCm6AT, CRTm6AYN6CTC, and CCm6AN7TGAG, and four novel serovar-specific motifs, GRTm6AN8TTYG, GAm6ACN7GTA, GAAm6ACY, and CAAm6ANCC. No changes in the percentage of methylated sites were observed after immersion in pepper except for BATGCm6AT, which increased. We cloned one nucleotide mutation, a deletion in mazG, into S. Tennessee, and characterized its phenotype using the Biolog' system. Growth of the wild type (WT) strain was comparable or slightly better than that of the mutant under most conditions tested, but the mutant was better able to utilize a small number of substrates including the carbon sources methyl pyruvate and D-lactic acid methyl ester. Together, these results suggest that DNA mutations and methylation may play a role in the adaptation and survival of S. enterica in LMFs, but more work is needed.