Construction of deletion mutants
4.2.3 Phage-complementation of clean-deletion mutants
Phage complementation was used in order to show that∆fliO,∆fliP,∆fliQand∆fliR clean-deletion strains can be complemented by introduction of the respective wild-type genes. Phage P22 grown on fliO−, fliP−, fliQ− and fliR− strains respectively, was used for infection of∆fliO, ∆fliP, ∆fliQas well as ∆fliRclean-deletion strains as described in Materials and Methods.
study. The table compares the motility of the different strains in presence and absence of tetracycline (Tc).
STRAIN GENOTYPE MOTILITY -Tc MOTILITY +Tc
TH10411 fliO6650::tetRA (after aa5) non-motile non-motile TH10412 fliO6651::tetRA (after aa121) non-motile ++
TH10413 fliP6652::tetRA (after aa5) non-motile non-motile TH10414 fliP6653::tetRA (after aa241) non-motile non-motile TH10415 fliQ6654::tetRA (after aa5) non-motile non-motile TH10416 fliQ6655::tetRA (after aa85) non-motile non-motile TH10417 fliR6656::tetRA (after aa5) non-motile non-motile TH10418 fliR6657::tetRA (after aa260) +++ + TH10419 ∆fliO6658::tetRA (∆aa6-121) non-motile non-motile TH10420 ∆fliP6659::tetRA (∆aa6-241) non-motile non-motile TH10421 ∆fliQ6660::tetRA (∆aa6-85) non-motile non-motile TH10422 ∆fliR6661::tetRA (∆aa6-260) non-motile non-motile
TH10548 ∆fliO6708 (∆aa6-121) non-motile N/A
TH10549 ∆fliP6709 (∆aa6-241) non-motile N/A
TH10550 ∆fliQ6710 (∆aa6-85) non-motile N/A
TH10551 ∆fliR6711 (∆aa6-260) non-motile N/A
As an example, the phage complementation assay of clean-deletion strain TH10548 (∆fliO) is displayed in Figure 4.5. Briefly, TH10548 was infected with phage P22 grown onfliO−,fliP−,fliQ− andfliR− strains respectively and incubated overnight at 37 °C. When the phage introduced wildtype fliO in TH10548 (∆fliO) cells, the particular infected cell regained motility. Since the phage, that introduced the wildtypefliOcannot insert in the bacterial chromosome and thus cannot repli-cate, only the originally infected mother cell is motile and swarms outwards, while leaving a trail of non-motile daugher cells behind. The non-motile daughter cells of this particular cell form visible colonies. As displayed in Figure 4.5, the clean dele-tion strain TH10548 (∆fliO) could be complemented by phage P22 grown on strains harboring wildtype fliO. The phage grown on the fliO− strain could not
comple-!fliO x
! fliO
-! fliP
-! fliQ
-! fliR -no !
FIGURE 4.5: TH10548 (∆fliO) phage complementation assay. TH10548 (∆fliO) was infected with phage P22 grown onfliO−,fliP−,fliQ−andfliR−strains respectively, and streaked on motility plates.
After overnight incubation, regained motility of TH10548 (∆fliO) cells could be observed as trails of non-motile daughter cells. Details of the process are discussed in the text.
ment the∆fliOstrain for obvious reasons. Furthermore, one can see that the clean deletion offliOis non-polar on downstream genes, since even the phages grown on fliP−, fliQ− and fliR− strains respectively, were able to complement the∆fliOclean deletion as shown in Figure 4.5. The same results were obtained by phage comple-mentation with TH10549 (∆fliP6709), TH10550 (∆fliQ6710) and TH10551 (∆fliR6711), showing that the clean deletion mutations of∆fliO,∆fliP,∆fliQand∆fliRrespectively, are non-polar and can be complemented by introduction of the respective wildtype genes (data not shown).
4.3 Discussion
In this study several strains, harboring tetRAinsertions infliO, fliP,fliQand fliR were constructed. ThetetRAinsertions at the 5’-start and 3’-end offliO,fliP,fliQand fliRrespectively, provide a useful tool for the subsequent construction of chromo-somally expressed N-terminal or C-terminal fusion proteins, as shown later in this work. For instance, the 3’-endtetRA-insertion strains were used for construction of C-terminal YFP fusions.
We showed that the tetRA-insertions at the 5’-start and 3’-end of fliO, fliP, fliQ and fliR respectively, were non-motile in the absence of tetracycline on motility plates with the exception of TH10418 (tetRAat the 3’-end offliR). This result might be explained with the presumption, that insertion of tetRA-cassettes in fliO, fliP, fliQandfliRrespectively, prevent correct translation of the polycistronic mRNA of fliLMNOPQR. Since fliR is the last gene in this operon, the insertion of the tetRA-cassette at the end offliRhas presumably no effect on the upstream genes.
In the presence of tetracycline, the genes of the tetRAcassette are induced. The tetRAgenes are transcribed in opposite direction, whereastetRreads out of the 5’-end andtetAreads out of the 3’-end. It is likely, that the transcription of thetetRA genes results in polar effects on the transcription of the up- and downstream genes and consequently, most of the tetRA-insertions at the 5’-start or 3’-end are non-motile. Surprisingly, the tetRA-insertions at the 3’-end of fliO (TH10412) and fliR (TH10418) are slightly motile, as shown in Figure 4.4. In the case of TH10418 one can argue that expression of the tetRA genes can only interfere with transcription of fliR and since the tetRA-insertion is located at the 3’-end, some functional FliR protein might be made. If this is the case and if only a few copies of functional FliR protein are necessary, this might be the explanation for the observed slightly motile phenotype. In the case of TH10412, the insertion of thetetRA-cassette at the 3’-end offliOmost likely interferes withfliOtranscription, but might allow correct expres-sion of the downstream genesfliP, fliQand fliR. In the presence of tetracycline, the
tetRAgenes are highly induced and sincetetAreads out of the 3’-end, the expression oftetAmight result in an over-expression of the downstream genesfliP, fliQandfliR and thus leading to the observed slightly motile phenotype, if expression offliOis not essential and can be complemented by over-expression offliP, fliQandfliR.
Furthermore, we showed thatfliO, fliP,fliQandfliRrespectively are essential for motility ofS. typhimurium, which confirms the results of Minamino and Macnab (56).
The clean-deletion strains constructed in this study showed no motility, but could be complemented by phage complementation, as well as by introduction of plasmids expressing a wildtype version of the respective deleted gene (Koushik Paul, unpub-lished data). Interestingly, over-expression offliPin the ∆fliObackground was able to complement the loss offliO, which leads to a motile phenotype (Koushik Paul, unpublished data). This result is consistent with the observed motile phenotype of thetetRAinsertion strain at the 3’-end offliO(TH10412) in the presence of tetracy-cline as described above.
In addition, this work shows that clean deletions of ∆fliO, ∆fliP, ∆fliQ and ∆fliR were non-polar by complementing the deletion strains using phage P22 grown on fliO−, fliP−, fliQ− and fliR− strains respectively. This is an important finding, since it allows the use of the∆fliO,∆fliP,∆fliQand∆fliRclean deletion strains for comple-mentation studies, like the verification of the functional expression of plasmid-based fliO,fliP,fliQandfliRconstructs.