DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA)

Abstract

Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam+) and GM33 (Δdam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA+ strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ΔgidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation caused by constitutive versus overproduction of Dam, as well as possible conformation of DNA influence the expression of act and gidA genes in A. hydrophila SSU. Our results indicate that the act gene is under the control of both Dam and GidA modification methylases, and Dam regulates Act production via GidA.

Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 – characterization of their genomes, proteomes and DNA methyltransferases

“…To our knowledge, it is also the first description of an active bacteriophage-carrying MTase which methylates adenine residues in many various contexts. So far, enzymes with such an extremely relaxed substrate specificity have been only found in defective prophages and phage-related elements (Drozdz et al, 2012;Fang et al, 2012). Like in the case of other non-specific adenine MTases, ARM81ld_p56 activity was not detected in the lysogenic strain.…”

“…Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis (Heithoff et al, 1999;Julio et al, 2001;Robinson et al, 2005). It has also been shown that the dam gene is essential for the viability of A. hydrophila SSU and that M. AhySSUDam is involved in A. hydrophila pathogenesis (Erova et al, 2006a(Erova et al, , b, 2012. The putative Dam-like protein of Aeromonas sp.…”

RNA modification enzymes encoded by the gid operon: Implications in biology and virulence of bacteria

“…In their study, a gidA deletion mutant was attenuated in mice due to a significant reduction in cytotoxic and hemolytic activity [58]. In a follow up study, Erova et al suggest act is under the regulatory control of both GidA and DNA adenine methyltransferase (Dam) and Dam regulates Act production via GidA [68]. Overall, these studies suggest GidA is part of a major regulatory pathogenic mechanism in Gram-negative bacteria.…”

“…In Myxococcus xanthus, deletion of gidA did not result in loss of motility as seen in other bacteria, but gidA deletion mutants were deficient in fruiting body development after several generations [13]. Finally, GidA was shown to significantly affect virulence in the emerging human pathogen Aeromonas hydrophila [58,68]. An initial study by Sha et al suggests GidA regulates the most potent virulence factor of A. hydrophila, the cytotoxic enterotoxin gene (act).…”

Modulation of host immune defenses by Aeromonas and Yersinia species: convergence on toxins secreted by various secretion systems

“…Of the 9, synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1) were knocked down in murine macrophages and HT-29 epithelial cells (using small inhibitory RNA), with the former two resulting in reduced induction of apoptosis following Act exposure (Galindo et al, 2006a,b). Interestingly, we also observed that DNA adenine methyltransferase (Dam) and Glucose inhibited division protein (GidA) both work to positively influence act gene expression and its associated hemolytic activity (Erova et al, 2012). Interestingly, the Gram-positive pathogen, Staphylococcus aureus , also secretes a potent, pro-apoptotic enterotoxin, the superantigen enterotoxin B.…”