The role of Dam methylation in the lifecycle of bacteriophage 933W
Bacteriophage 933W is a member of the lambdoid family of which phage lambda is the best studied. Lambda can exist either in the lytic or lysogenic state. In the lytic state the virus infects the bacterial cell and makes many progeny which are released on cell rupture. In the lysogenic state the virus is integrated in the host chromosome where it remains dormant until an environmental signal causes it to eject from the chromosome and enter the lytic cycle.
933W is a lysogenic phage from enterohemorrhagic Escherichia coli O157:H7 and carries the stx2A genes which produce Shiga toxin. Unlike lambda, 933W also has a dam gene encoding a Dam methyltransferase which produces 6-methyl adenine in the sequence GATC. The E. coli host also has a dam gene producing a Dam methyltransferase with the same sequence specificity. Dam methylation has various functions in E. coli (Marinus and Lobner-Olesen, 2014) but nothing is known about the its role in phage 933W (and other stx phages).
To study the role of dam methylation, the stx2AB and dam genes were removed from 933W and all experiments used the non-pathogenic laboratory strain E. coli K-12 as the host. 933W and 933W dam were plated on E. coli K-12 wildtype and a dam derivative to determine plaque morphology. Clear plaques on the host strain indicate the phage is in the lytic mode (- in Table 1) while turbid plaques indicate a lysogenic mode (+ in Table 1).
Table 1. Plaque formation of 933W and 933W dam on wildtype and dam strains of E coli K-12.
| Phage | dam+ host | dam- host |
| 933W wild | + | - |
| 933W dam | + | + |
| 933W dam p35 | - | - |
933W forms turbid plaques on the wildtype host but clear plaques on the dam host. The basis for the inability of 933W to form lysogens in a dam host is not known but may be due to the SOS-induced state of dam cells. 933W dam forms turbid plaques on both the wildtype and dam hosts. In a separate experiment about 50% of cells in plaques of 933W on the wildtype were lysogens while greater than 95% of 933W dam on wildtype and dam hosts were lysogens.
Within the turbid plaques of 933W dam on the dam host were secondary tiny clear plaques. Four independently isolated of these clear plaque formers were isolated and shown to have mutations in the phage p35 gene. The p35 gene is next to the dam gene on the phage map but its function is unknown. The p35 mutations suppress the phenotype of 933W and 933W dam as the double mutant phage (933W dam p35) forms clear plaques on wildtype and dam hosts (Table 1).
Lysogens of a particular phage are resistant to infection by the same phage. Lysogens produce repressor protein (cI repressor) which binds to promoter sequences preventing lytic development. CI repressor binds to the same promoters in the incoming phage preventing lytic development. All 933W phages used here have the same cI repressor.
933W dam p35, however, forms clear plaques on 933W lysogens and turbid plaques on 933W dam lysogens (Table 2).
Table 2. 933W dam p35 plaque morphology on wildtype and dam lysogens.
| Phage | dam+ lysogen | dam- lysogen |
| dam p35 | - | + |
In the wildtype host 933W dam p35 is able to overcome the effect of repressor and produce clear plaques but the opposite occurs in dam lysogens (Table 2).
Although the above data do not allow a conclusion as to the role of dam methylation, it does suggest that the dam and p35 gene products affect the lysis/lysogeny balance.