Igls Poster Results

RESULTS

1. Construction of deletion mutant of the HsdS subunit

We have constructed a deletion mutation of the hsdS gene between the XmnI and NruI restriction sites such that the reading frame of the gene is maintained and only amino acids 161-190 from the central-conserved, repeated amino acid sequence are removed (Figure 2). This mutant has a Res^- Mod^- phenotype (Table 2) and when co-produced with HsdM the deleted HsdS subunit was found in the pellet fraction (Figure 3) (the test used to demonstrate protein-protein interactions [4,7] ). This deletion prevents subunit assembly, possibly because of the lack of the important domain required for HsdS-HsdM interactions.

2. PCR-based mutagenesis of the central conserved region of the hsdS gene

To investigate further the importance of the central conserved region of HsdS for subunit assembly, we have used PCR-based misincorporation mutagenesis, in the presence of 0.1mM and 0.5mM Mn²⁺ to produce a collection of mutations [5]. PCR misincorporation mutagenesis was carried out using primers shown in Figure 2 and the PCR product was inserted back into the EcoRI-NcoI digest of pJS491. Phenotypes of resulting plasmids were analysed in competition assay and interesting mutants were sequenced.

3. Complementation (competition) analysis

Complementation analysis between the plasmids carrying the PCR fragments and plasmid pKF650 (which carries the hsd genes of the EcoRl24II R-M system) was used to analyse the restriction and modification phenotype of the transformants. (This competition assay has been used previously to discriminate DNA-binding mutations from subunit assembly mutations, and all mutants with a Res^- Mod^- phenotype were found to give no competition with the alternate HsdS subunit produced from pKF650 and were proposed to be subunit assembly mutants [6] ). From a total of two hundred transformants 71 mutants were obtained. Of these mutants 61 were found to be Res^- mutants; however, because these mutants showed no competition in the complementation assay which suggested that they are unable to assemble with HsdM and HsdR and may be unfolded (Table 1). The classical mutations within hsdS genes were always found to produce both restriction and modification-deficient phenotype. Two mutants (Nos. 72 and 123) were found to have a Res^- Mod⁺ phenotype. These are non-classical mutations, which cannot arise from an alteration of the DNA-binding properties of the HsdS subunit since both produce a fully active DNA methyltransferase (Table 2 and 3).

4. Solubility of mutant HsdS subunits

This test is based on the evidence that HsdM is found to be soluble and HsdS insoluble, in bacterial lysates. In the presence of the co-produced HsdM subunit, HsdS is found in the soluble fraction as part of an active methylase [7]. Mutants were sub-cloned into the plasmid pJS4M, replacing the wild-type hsdS with the respective mutant genes and tested for in vivo modification function (Table 3) and for solubility (Figure 3A). The deleted HsdS subunit (HsdSDXN) was found in the pellet fraction, while the HsdS72 and HsdS123 subunits are soluble, which is not surprising considering the Mod⁺ phenotype.

5. Western blot analysis

Although over-produced HsdS and HsdM subunits were clearly detected on SDS-PAGE, the ECL Western blotting method was used to confirm it. The individual subunits were identified using polyclonal antibodies raised in rabbit against purified M.EcoRl24I. The immune complexes were visualised by chemiluminscence using horseradish peroxidase-labelled secondary antibodies. The assay proved that protein band in position of HsdS present in some fractions, in which no wild-type HsdS was expected, is an unknown protein from the host strain JM109(DE3) (Figure 3B).

6. Localization of the mutation in hsdS72 and hsdSl23

DNA sequence analysis of these mutations revealed two changes. One of them (T->G) at position 2608 is a silent mutation which does not change the amino acid. Further sequence analysis of the wild-type hsdS gene showed that this change is also present in the wild-type gene and must be a mistake in the original sequence. In fact both mutant hsdS genes resulted from a single point mutation. HsdS72 has a mutation T->C, at position 2594 which results in a change of Trp212 to Arg. Mutation A->T at position 2512 in hsdSl23 results in a change of Lysl84 to Asn.

Table 1. Frequency of mutations following PCR--misincorporation mutagenesis.

Concentration of Mn²⁺	All mutations	Res- mutations
0.lmM Mn²⁺	20%	17%
0.5mM Mn²⁺	51%	44%

Table 2. Restriction-modification phenotype of mutant hsdS genes in a competition assay with EcoRl24II.

Plasmid(s)	DNA specificity	Restriction (e.o.p.)^a	Modification (e.o.p.)^a	Phenotype
pKF650	EcoRl24II / EcoRl24I	0.002 / 1.0	0.4 / 0.002	Res⁺ Mod⁺ Res^-Mod^-
pKF650+pJS491	EcoR124II / EcoR124I	0.04 / 0.0004	1.0 / 1.0	Res⁺ Mod⁺ Res⁺ Mod⁺
pKF650+ pJSDXN	EcoR124II / EcoR124I	0.001 / 0.8	1.0 / 0.001	Res⁺ Mod⁺ Res^-Mod^-
pKF650+pJS72	EcoR124II / EcoR124I	0.2 / 0.5	1.0 / 0.7	Res^- Mod⁺ Res^-Mod⁺
pKF650+ pJS123	EcoR124II / EcoR124I	0.2 / 0.8	1.0 / 0.4	Res^- Mod⁺ Res^-Mod⁺

^a efficiency of plating of corresponding lambda phage.

All assays were carried out in JM109(DE3) in the absence of IPTG, the background level of T7 RNA polymerase has been found to be sufficient for restriction and modification activity.

Plasmid pKF650 carries the hsd genes for the EcoRl24II R-M system; plasmid pJS491 carries the wild-type hsdS gene of EcoRl24I under the control of the T7 g10 promoter; plasmid pJSDXN is the derivative of pJS49l with a 29-aa deletion between XmnI and NruI; plasmids pJS72 and pJS123 are the equivalents of pJS491, but carrying the hsdS72 and hsdS123 mutations respectively.

Table 3. In vivo methylation of bacteriophage lambda by the mutant M.EcoRl24

Plasmid	Modification (e.o.p.)^a	Phenotype
pJS4M	0.9	Mod⁺
pJS72M	0.9	Mod⁺
pJS123M	0.8	Mod⁺
pJSDXNM	0.001	Mod^-

^a efficiency of plating of lambda phage (produced on the tested transformants) on E.coli C[R124] (Res⁺ Mod⁺) relative to E. coli C (Res^- Mod^-) reflects the level of in vivo methylation. All assays were carried out in JM109(DE3) in the absence of IPTG.

Plasmid pJS4M carries the hsdM and hsdS genes of EcoRl24I both under the control of the T7 gl0 promoter; plasmid pJS72M, pJS123M and pJSDXNM are the equivalents of pJS4M but with the hsdS72, hsdSl23 and hsdSDXN mutant genes respectively.

Figure 1.

Schematic model of the interaction between anti-restriction proteins Ard and Type I R-M systems. Mechanism of this interaction is based on the sequence similarity of conserved elements (closed boxes) of Ard proteins (anti-restriction domain, box A) and HsdS subunits (Argos repeat-like sequences [3], boxes A1 and A2) and their competition for the interaction with HsdM subunits [2].

Figure 2. Location of non-classical mutations in hsdS

Domain organisation of the HsdS subunit of EcoR124I. The conserved domains of the HsdS subunit are shown as shaded regions, the arrows under these domains represent the repeated amino acid sequences [4]. The smaller arrows above the amino acid sequence show the two repeated amino acid sequences present in EcoR124I, which are subject to unequal crossover to produce the novel DNA specificity present in EcoR124II. The box, around the amino acid sequence, is the region of DNA, between the XmnI and NruI restriction sites, deleted in the mutant hsdSDXN. The positions of hsdS72 and hsdSl23 mutations are indicated.