ABSTRACTVibrio choleraegenome sequences were analyzed for variation in thertxAgene that encodes the multifunctional autoprocessing RTX (MARTX) toxin. To accommodate genomic analysis, a discrepancy in the annotatedrtxAstart site was resolved experimentally. The correct start site is an ATG downstream fromrtxCresulting in a gene of 13,638 bp and deduced protein of 4,545 amino acids. Among the El Tor O1 and closely related O139 and O37 genomes,rtxAwas highly conserved, with nine alleles differing by only 1 to 6 nucleotides in 100 years. In contrast, 12 alleles from environment-associated isolates are highly variable, at 1 to 3% by nucleotide and 3 to 7% by amino acid. The difference in variation rates did not represent a bias for conservation of the El TorrtxAcompared to that of other strains but rather reflected the lack of gene variation in overall genomes. Three alleles were identified that would affect the function of the MARTX toxin. Two environmental isolates carry novel arrangements of effector domains. These include a variant from RC385 that would suggest an adenylate cyclase toxin and from HE-09 that may have actin ADP-ribosylating activity. Within the recently emerged altered El Tor strains that have a classicalctxBgene, a mutation arose inrtxAthat introduces a premature stop codon that disabled toxin function. This null mutant is the genetic background for subsequent emergence of thectxB7allele resulting in the strain that spread into Haiti in 2010. Thus, similar to classical strains, the altered El Tor pandemic strains eliminatedrtxAafter acquiring a classicalctxB.IMPORTANCEPathogen evolution involves both gain and loss of factors that influence disease. In the environment, bacteria evolve rapidly, with nucleotide diversity arising by genetic modification. Such is occurring withVibrio cholerae, exemplified by extensive diversity and unique variants of thertxA-encoded multifunctional autoprocessing RTX (MARTX) toxin among environment-associated strains that cause localized diarrheal outbreaks and food-borne disease. In contrast, seventh pandemic El TorV. choleraestrains associated with severe diarrhea have changed minimally until the altered El Tor emerged as the most frequent cause of cholera, including in the 2010 Haiti epidemic. These strains have increased virulence attributed to a new variant of the major virulence factor, cholera toxin. It is revealed that these strains also have an inactivated MARTX toxin gene. A similar inactivation occurred during classical cholera pandemics, highlighting that evolution of El Tor cholera is following a similar path of increased dependence on cholera toxin, while eliminating other secreted factors.
Long-Read-Based Genome Sequences of Pandemic and Environmental Vibrio cholerae Strains