Abstract
Cronobacter species are a group of foodborne pathogenic bacteria that cause both intestinal and systemic human disease in individuals of all age groups. Little is known about the mechanisms that Cronobacter employ to survive and persist in foods and other environments. Toxin–antitoxin (TA) genes are thought to play a role in bacterial stress physiology, as well as in the stabilization of horizontally-acquired re-combinatorial elements such as plasmids, phage, and transposons. TA systems have been implicated in the formation of a persistence phenotype in some bacterial species including Escherichia coli and Salmonella. This project’s goal was to understand the phylogenetic relatedness among TA genes present in Cronobacter. Preliminary studies showed that two typical toxin genes, fic and hipA followed species evolutionary lines. A local database of 22 TA homologs was created for Cronobacter sakazakii and a Python version 3 shell script was generated to extract TA FASTA sequences present in 234 C. sakazakii genomes previously sequenced as part of Center for Food Safety and Applied Nutrition’s (CFSAN) GenomeTrakr project. BLAST analysis showed that not every C. sakazakii strain possessed all twenty-two TA loci. Interestingly, some strains contained either a toxin or an antitoxin component, but not both. Five common toxin genes: ESA_00258 (parDE toxin-antitoxin family), ESA_00804 (relBE family), ESA_01887 (relBE family), ESA_03838 (relBE family), and ESA_04273 (YhfG-Fic family) were selected for PCR analysis and the primers were designed to detect these genes. PCR analysis showed that 55 of 63 strains possessed three of these genes Sequence analysis identified homologs of the target genes and some of the strains were PCR-negative for one or more of the genes, pointing to potential nucleotide polymorphisms in those loci or that these toxin genes were absent. Phylogenetic studies using a Cronobacter pan genomic microarray showed that for the most part TAs follow species evolutionary lines except for a few toxin genes possessed by some C. malonaticus and C. universalis strains; this demonstrates that some TA orthologues share a common phylogeny. Within the C. sakazakii strains, the prevalence and distribution of these TA homologs by C. sakazakii strain BAA-894 (a powdered infant formula isolate) followed sequence-type evolutionary lineages. Understanding the phylogeny of TAs among the Cronobacter species is essential to design future studies to realize the physiological mechanisms and roles for TAs in stress adaptation and persistence of Cronobacter within food matrices and food processing environments.
Highlights
Toxin–antitoxin (TA) systems are small genetic elements found on plasmids, phage genomes, and chromosomes of many bacterial and archaeal species [1,2,3,4]
There were two phage TA homologs [encoding for YeeV toxin protein and YkfI toxin protein, prophage TAs from the C. sakazakii strain 29544T] that were represented on the microarray and were from C. dublinensis and C. malonaticus strain LMG2326T
Molecular clock analysis demonstrated that C. sakazakii strains possibly descended from an ancestral strain(s) that might have acquired TA genes at distinct evolutionary incidences that correlated with occurrence and evolution of sequence type (ST) lineages
Summary
Toxin–antitoxin (TA) systems are small genetic elements found on plasmids, phage genomes, and chromosomes of many bacterial and archaeal species [1,2,3,4]. They are usually observed as bi-cistronic gene pairs with the antitoxin gene preceding the toxin gene. When bacteria encounter a stressful environment, the antitoxin gets degraded by host proteases, such as Lon and ClpXP; which releases the toxin to bind to mRNA transcripts, halting essential transcription of key genes involved in the physiological processes like transcription/translation of critical cellular processes, cell division, DNA replication, ATP synthesis, mRNA stability, or cell wall synthesis [5]. A recent report by Goormaghtigh et al [8] showed that the direct link between induction of type II TA systems and persistence brought on by antibiotics might not be as straightforward as was once thought in these cells
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