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 (∆dam) 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 ∆gidA 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.

Introduction

Among various Aeromonas species, A. hydrophila is an aquatic environmental and food-borne microorganism which poses a health risk (Edberg et al., 2007). A diarrheal isolate SSU of A. hydrophila studied in our laboratory is involved in human infections, and its major virulence factor, the cytotoxic enterotoxin (Act), leads to septicemia, gastroenteritis and mortality in a mouse model (Chopra and Houston, 1999, Sha et al., 2001, Sha et al., 2002). We reported that a glucose-inhibited division protein, GidA, modulated the expression of the act gene (Sha et al., 2004).

GidA protein was first described in the Escherichia coli K12 strain, and disruption of the gidA gene affected cell division when grown in a medium containing glucose by interrupting chromosomal replication, resulting in a cell elongation phenotype (von Meyenburg et al., 1982). Further studies showed that GidA is a flavin adenine dinucleotide (FAD) binding enzyme (White et al., 2001) and also a tRNA modification methylase that catalyzes the addition of the carboxymethylaminomethyl group onto the C5 carbon atom of uridine at position 34 (U₃₄) of RNAs (Urbonavicius et al., 2005, Yim et al., 2006). It was found that the post-transcriptional modification of tRNA represents a significant element controlling gene expression (Gustilo et al., 2008).

The deletion of a gidA gene attenuated the pathogenesis of some bacteria, such as Streptococcus pyogenes, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium (Cho and Caparon, 2008, Gupta et al., 2009, Shippy et al., 2011). In particular, it was noted that gidA gene mutant had a nearly normal global transcription profile, when compared to that of parental S. pyogenes strain, but the mutant produced significantly reduced levels of multiple virulence factors due to impaired translational efficiencies (Cho and Caparon, 2008). The gidA mutation in a plant pathogen P. syringae led to pleiotropic effects, resulting in diverse phenotypic traits, swarming, presence of fluorescent pigment and virulence, which indicated a possible regulatory role for GidA in moderating translational fidelity (Kinscherf and Willis, 2002).

On the other hand, DNA adenine methyltransferase (Dam) methylates the adenine base of 5′-GATC-3′ specific sites and is a global gene regulator modifying DNA. Thus, Dam plays an important role in controlling various processes in prokaryotic cells, such as transcriptional regulation, mismatch repair, host–pathogen interactions, and binding of the replication initiation complex to the methylated origin of replication (oriC) site (Casadesus and Low, 2006, Chatti and Landoulsi, 2008, Low and Casadesus, 2008, Marinus and Casadesus, 2009). It is known that initiation of replication of the chromosome occurs at a unique site, namely the oriC. Among the promoters possibly involved in the transcriptional activation of replication initiation included those for the gidAB genes, as transcription of gid genes was needed for activation of oriC (Bates et al., 1997). Further, regulation of DNA methylation activity through promoter methylation and compatibility between methylation of a promoter with its active transcription have been reported (Barnard et al., 2004, Marinus and Casadesus, 2009). Dam methylation can control promoter transcription, and transcriptional repression by Dam appears to be more common than transcriptional activation.

Our previous results showed that overproduction of Dam in A. hydrophila SSU increased Act-associated biological activities; however, a decrease in such activities was noted in the gidA gene mutant of the corresponding parental strain which produced a constitutive level of Dam (Erova et al., 2006a, Erova et al., 2006b). These data indicated that the extent of DNA methylation which was governed by the amount of Dam present dictated the levels of GidA and Act in A. hydrophila SSU. Dam and GidA proteins are highly conserved in many prokaryotes, and because the dam gene is essential for the viability of the A. hydrophila SSU strain, but not of E. coli, this fact allowed us to use E. coli K12 as a model to address the hypothesis regarding act gene regulation by both enzymes. We show data which further our understanding on the cross-talk between the decisive regulators of gene expression, namely dam and gidA, on Act production in A. hydrophila SSU. Our previous data indicated that Act is the most potent virulence factor among the three enterotoxins of A. hydrophila SSU, and GidA and Dam are involved in its control (Erova et al., 2006a, Sha et al., 2004). In the present study, we explored how these regulatory proteins, Dam and GidA, influence Act production. These studies are important, as modulation of bacterial virulence genes in general could be under the control of Dam and GidA and needs further investigation.