Characterization of type IV pili gene deletion mutants

difficult to detect and obviously weaker compared to the expression of pilB1. This was inferred from the cycle of threshold (CT) values of both genes (CT = 21 for pilB1 and CT = 31 for pilB2) (Figure 20B). Even though no absolute quantification of transcript amounts was performed, the CT values of both genes differed by ten cycles and therefore clearly indicated higher expression of pilB1, while pilB2 was expressed only weakly.

Figure 20 Transcription analysis of the Hbt. salinarum R1 type IV pili system assembly ATPase encoding genes. A, Relative transcriptional quantification of the assembly ATPase encoding genes of the archaellum (flaI) and the putative type IV pilus biogenesis systems pil-1(pilB1) and pil-2(pilB2) as well as the constitutively expressed ferredoxin gene (fdx). The bars represent the fold change of gene expression shown in base 2 logarithmic scale in adherent cells compared to the planktonic state, which is defined by the baseline. B, Average CT values of the taget genes fdx, flaI, pilB1 and pilB2 detected in adherent cells.

In summary, cotranscription of the genes pilB1 and pilB2 with downstream genes from the pil-1 and pil-2 loci, respectively, was demonstrated. The transcription patterns of both genes, i.e.

higher expression in adherent cells of Hbt. salinarum R1, prompted a role in the adhesion.

4.2.4. Characterization of type IV pili gene deletion mutants

Gene deletion mutants were characterized to test the involvement of the various Halobacterium salinarum R1 type IV pili systems (T4P) in adhesion. These mutants lacked flaI or flaI together with pilB1, respectively, a combination of the three genes flaI, pilB1 and pilB2. The gene deletion mutants were provided by Lucia Vidakovic (Master thesis L. Vidakovic, 2014). These mutants were generated using a pop-in/pop-out strategy, as described previously (Koch &

Oesterhelt, 2005).

Validation of the gene deletion mutants was carried out by PCR employing oligonucleotides flanking the genomic sites of the genes which were intended for deletion, i.e. flaI, pilB1 and pilB2. Mutants with a successful deletion yielded a shorter PCR product by about the length of

genes (∆flaI/∆pilB1) as well as a combination of all three Halobacterium T4P ATPase genes (∆flaI/∆pilB1/∆pilB2) were verified (Figure 21).

Figure 21 Verification of Hbt. salinarum R1 parental, ΔflaI single deletion, ΔflaI/ΔpilB1 double deletion and ΔflaI/ΔpilB1/ΔpilB2 triple deletion mutant strains. PCRs using genomic DNA isolated from the respective strains as template were carried out with oligonucleotides flanking the flaI, pilB1 and pilB2 genomic regions (listed in section 2.1.4). The absence of flaI, pilB1 or pilB2 genes leads to a reduced fragment size of 1.2 kbp, 1.5 kbp and 1.6 kbp respectively. Control reactions using water as template were performed.

Growth of the Hbt. salinarum R1 parental strain as well as the ∆flaI, ∆flaI/∆pilB1 and the

∆flaI/∆pilB1/∆pilB2 mutant strains was compared by monitoring the increase of the optical density at a wavelength of 600 nm (OD600) over a time course of about 100 h.

Figure 22 Growth and adhesion of the Hbt. salinarum R1 parental strain in comparison to the gene deletion mutants. A, Growth of Hbt. salinarum R1 parental strain (black squares) and the ΔflaI (black diamonds), ΔflaI/ΔpilB1 (grey triangles) and the ΔflaI/ΔpilB1/ΔpilB2 (grey dots) mutants. Diagram shows semilogarithmic plot of the optical density at 600 nm (OD600) as a function of incubation time in hours. B, Fluorescence-based adhesion assay of the Hbt. salinarum R1 parental strain and ΔflaI, ΔflaI/ΔpilB1 and the ΔflaI/ΔpilB1/ΔpilB2 mutants. Relative adhesion of the mutant strains in relation to the parental strain after 15 days of growth is illustrated.

The resulting growth curves did not show notable differences (Figure 22A). All four strains entered a logarithmic growth phase after 16 h, which lasted until the 48 h point of time, when the curves start flattening. A plateau was reached after 80 h of incubation, with no noteable further increase of OD600. The final optical density was similar in all four strains. From these results no negative impacts on growth of the cells appeared as consequence of the mutations.

Nevertheless, differing adhesion properties to plastic surfaces were observed with the four strains, in a fluorescence-based adhesion assay (Figure 22B). The parental strain and the ∆flaI mutant exhibited strong adhesion, whereas the double and triple gene deletion mutants both displayed relative adhesion signals of only 20% in comparison to the parental strain (Master thesis L. Vidakovic, 2014).

Differences with regard to motility were observed in swimming motility assays of the four strains, using semi-solid agar plates, which were inoculated in the centre with a droplet of cell suspension of the different strains. While the parental strain displayed motility and a swarming radius of about 2.5-3.0 cm after three days of static incubation, all three deletion mutant strains did not spread on the surface of the semi-solid agar plates (Figure 23A). Planktonic cells of all four strains had a regular rod-shaped cell morphology, as observed by light microscopy (Figure 23B). Only the parental strain was motile and freely swimming, whereas no motility was observed for planktonic cells of any of the three gene deletion strains.

Adhesion of the cells to glass coverslips and biofilm formation on plastic surfaces was investigated by phase contrast microscopy (PCM) and confocal laser scanning microscopy (CLSM). Extensive adhesion was only observed in the case of the Hbt. sainarum R1 parental and the ∆flaI strain (Figure 23C). In contrast, only few attached cells and cell accumulations were visible with the double and triple gene deletion strains. The parental and ∆flaI strains both formed biofilms on plastic surfaces, although there existed apparently differences regarding the biofilm architecture (Figure 23D). The biofilms formed by the ∆flaI mutant appeared flat and dense, showing less microcolonies or tower-like structures. After 15d of incubation dense adherence of the ∆flaI cells over the surface was observed including the formation of 15 to 20 µm thick biofilms, whereas the other two mutant strains adhered only weakly to the plastic surface.

Figure 23 Characterization of the Hbt. salinarum R1 parental strain in comparison to the gene deletion mutants. A, Swimming assay of Hbt. salinarum R1 parental strain (R1) and the ΔflaI, ΔflaI/ΔpilB1 and the ΔflaI/ΔpilB1/ΔpilB2 mutants on semi solid agar plates. Scale bars: 1 cm. B, Corresponding phase contrast micrographs depicting planktonic cells of the four strains. C, Phase contrast micrographs of cells adhering to glass coverslips after 15 d of growth. B and C: 100x magnification, scale bars 10 µm. D, Corresponding CLSM projections depicting biofilms formed. Cells stained with acridine orange. Tilted top views (top) and side views (bottom) of biofilms grown for 15 d. 63x magnification, scale bars 30 µm.

Table 5 Overview of the characterization of the Hbt. salinarum R1 parental and gene deletion strains

Strain Motility Adhesion Biofilm

formation

Filament types*

R1 yes strong yes 2

∆flaI no strong yes 1

∆flaI/∆pilB1 no weak no 0

∆flaI/∆pilB1/∆pilB2 no weak no n.d.

*According to TEM analyses (Master thesis L. Vidakovic, 2014); n.d. not determined

In summary, in contrast to the motile parental strain strain, all three mutants investigated were non-motile, because ∆flaI leads to the deletion of the archaella. Nevertheless, the ∆flaI strain showed adhesion comparable to the parental strain as well as biofilm formation, but with altered architectures. In comparison, the ability to adhere and to form biofilms was strongly reduced upon additional deletion of the pilB1 gene in the ∆flaI/∆pilB1 mutant. Further deletion of pilB2 had no additional effects on adhesion in the ∆flaI/∆pilB1/∆pilB2 mutant.