Structural and functional analysis of PhspA by mutagenesis

To find out how the putative regulatory element (G3) is involved in the transcriptional regulation of hspA and whether there are other regulatory elements involved in the transcriptional regulation of hspA under heat shock conditions, PhspA has been further studied

by deletion analyses. A series of plasmids were constructed in which different deletions were introduced in the upstream sequence of hspA. The modified upstream regions of hspA were subsequently fused to the rtrpA-lacZ reporter gene. After integration of these plasmids into the attB site of DW4/3-1, the expression of lacZ under the control of the mutated PhspA was analysed.

2.4.3.1. Influence of the first 65 bp of hspA coding region on hspA expression

To elucidate whether the first 65 bp coding sequences of hspA are involved in the expression of hspA, plasmid pSH42 was constructed by cloning an EcoRI/BamHI restricted PCR fragment into the EcoRI/BamHI sites of pSH8. This PCR fragment carries the 225 bp upstream region of hspA, which was generated with the primer pair S4E and B4 using pSH1 as template.

Integration of pSH42 into the attB site of DW4/3-1 resulted in strain SH2822. Strains SH2805 (Fig. 2.14) and SH2822 (Fig. 2.20) showed the same ß-galactosidase activity after heat shock. This indicates that the first 65 bp coding sequence of hspA has no influence on hspA transcription. But this does not exclude this region to influence translation in appropriate strains.

2.4.3.2. The function of the G3 region

To analyse the contribution of G3 to hspA expression, mutants SH2816 and SH2820 that harbour deletions in the ectopically integrated hspA promoter region were constructed (Fig. 2.20). They were obtained by integration of the plasmids pSH29 and pSH30 into the attB site of the wild-type DW4/3-1 strain.

To construct pSH29, the upstream region of hspA from bp -379 to bp -212 was amplified with the primer pair E3 and C2 using pSH1 as template. The PCR product was cloned into the EcoRI/ClaI sites of pBR322 to generate pSH29A. Another DNA fragment ranging from bp –146 to bp +65 was generated with the primer pair C9 and B1 using pSH1 as template.

The resulting fragment was cloned into the ClaI/BamHI sites of pSH29A to generate pSH29B. The EcoRI-BamHI insert of pSH29B that ranges from bp -379 to bp +65 harbouring a deletion from bp -211 to bp -147 was subcloned into pSH8 to generate pSH29.

Plasmid pSH30 was constructed similarly. Using the primer pair E3 and C14, the upstream region of hspA from bp -379 to bp -224 was amplified and cloned into the EcoRI/ClaI sites of pBR322 to generate pSH30A. The sequence from bp -85 bp +65 was amplified using the primer pair C11 and B1 and cloned into the ClaI/BamHI sites of pSH30A resulting in pSH30B. The EcoRI/BamHI insert of pSH30B was subcloned into pSH8 to

generate pSH30, which contains a fragment ranging from bp -379 to bp +65 with a deletion that ranges from bp -223 to bp -86.

Fig. 2.20 shows that the maximum ß-galactosidase activity induced by heat shock in strain SH2816 is only 38% of that obtained in strain SH2804. In strain SH2816, most of the putative regulator binding region, G3, is deleted. This suggests the putative regulator(s) that binds to G3 to be transcription activator(s).

BamHI

Fig. 2.20. Transcriptional activity analysis of mutated PhspA. The deletion region of each construct is indicated in the figure. Soluble protein was isolated from strains SH2820, SH2816, SH2813, SH2811, and SH2822 before and 80 minutes after temperature up-shift from 28°C to 38°C. ß-galactosidase activity was determined as in 2.3.2.3. Strain SH2804 was used as a control.

Surprisingly, the heat shock induced maximum ß-galactosidase activity of strain SH2820 was reduced to only about 6% of that determined in strain SH2804 (Fig. 2.20). The different deletion effect between SH2820 and SH2816 suggests that a cis-acting element should exist just upstream of the -35 region in PhspA .

2.4.3.3. The putative regulator binding site(s) on G3

The DNA fragment G3 contains two inverted repeats (Fig. 2.6). One of them is located from bp -214 to bp -196 with the sequence: 5’-CCGCCTGN₅CAGGCGG-3’ or alternatively from bp -213 to bp -185 with the sequence: 5’-CGCCTGN₁₇CAGGCG-3’. The second one is located between bp -165 and bp -144 with the sequence: 5’-CCCTTN12AAGGG-3’. One might speculate that these inverted repeats are the binding sites for the transcription activator(s).

To analyse the influence of the inverted repeats of G3 on the transcriptional activation of PhspA, plasmids pSH24 and pSH26 were constructed in which the inverted repeat regions were deleted.

The upstream region of hspA from bp -379 to bp -187 was amplified using the primer pair E3 and C6 and cloned into the EcoRI/ClaI sites of pBR322 resulting in pSH24A. The region from bp -146 to bp +65 was amplified using the primer pair C9 and B1 and cloned into the ClaI/BamHI sites of pSH24A to generate pSH24B. The EcoRI-BamHI insert of pSH24B that renges from bp -379 to bp +65 containing a deletion ranging from bp -186 to bp -147 was subcloned into pSH8 resulting in pSH24.

In the same way, pSH26 was constructed that contains the region from bp -379 to bp +65 harbouring a deletion from bp -211 to bp -199. The primer pairs used for generating this fragment were E3 and C2 or C3 and B1, respectively.

Integration of plasmids pSH24 and pSH26 into the attB site of S. aurantiaca DW4/3-1 resulted in strains SH2811 and SH2813 (Fig. 2.20).

In strain SH2813, a part of the first inverted repeat was deleted. This led to about 55%

reduction of the heat induced maximum ß-galactosidase activity. In promoter mapping assay (2.3.3), deletion of the upstream sequence of hspA to bp -192 (SH2819) resulted in the same reduction (about 50%, Fig. 2.14).

Deletion of the second inverted repeat (SH2811) reduced the heat induced maximum ß–galactosidase activity to about 65%.

Neither of the two deletions (SH2811 and SH2813) decreased the maximum ß–galactosidase activity to the same extent as the deletion covering both inverted repeats (SH2816). If one assumes that there is no synergistic interaction between both inverted repeats, the maximum ß-galactosidase activity of strain SH2816 should be reduced to about 36% (product of the reduction in strains SH2811 and SH2813). This agrees quite well with the maximum ß-galactosidase activity measured in strain SH2816 (38%).