Related Experiment Video
Updated: Aug 23, 2026
DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments
Published on: January 27, 2016
Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence
Michael E Watson1, Justin Jarisch, Arnold L Smith
1Seattle Biomedical Research Institute, 307 Westlake, Suite 500, Seattle, WA 98109-5219, USA.
Abstract:
Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors.
Insights
Deoxyadenosine methyltransferase (dam) is crucial for DNA repair and virulence in Haemophilus influenzae. Dam mutants show impaired invasion and altered virulence, highlighting its regulatory role in bacterial pathogens.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Deoxyadenosine methyltransferase (Dam) methylase plays diverse roles in Gram-negative pathogens, including DNA repair, replication control, and gene regulation.
- Dam methylation influences gene expression, particularly virulence factors, through promoter methylation.
Purpose of the Study:
- To investigate the roles of Dam methylase in Haemophilus influenzae, focusing on DNA mismatch repair and virulence.
- To characterize dam mutants in H. influenzae strains Rd KW20, Strain 12, and INT-1.
Main Methods:
- Inactivation of the dam methylase gene (HI0209) in H. influenzae strains.
- Confirmation of Dam methylation absence using methylation-sensitive enzymes (DpnI, DpnII).
- Assessment of 2-aminopurine susceptibility, invasion assays (HBMECs, NCI-H292), intracellular replication, and infant rat model of infection.
Main Results:
- Dam mutants exhibited 2-aminopurine susceptibility, indicating a role in DNA mismatch repair, suppressed by mutS mutations.
- Invasion of host cells (HBMECs, NCI-H292) was significantly attenuated in all dam mutants.
- Virulence was reduced in vivo (infant rat model) for the INT-1 dam mutant, and intracellular replication was inhibited in the Strain 12 dam mutant.
Conclusions:
- Dam methylase is essential for H. influenzae virulence in a strain-dependent manner, affecting both in vitro and in vivo pathogenicity.
- Dam activity regulates gene expression, including virulence factors, and is involved in DNA mismatch repair in H. influenzae.
Related Concept Videos
01:25Inhibitors Of Virion Release
01:28Gene Regulation in Microbial Communities: Quorum Sensing
01:20Determinants of Bacterial Pathogenicity and Virulence