Estudios anteriores utilizando la metodología “Propensity Score”

adhere to surfaces (Fröls et al., 2012). Among the strains investigated were the type strain DSM3754T_{, the three closely related wild type strains R1, NRC-1, PHH1 and the PHH1}

derivative PHH4, as well as the two natural isolates strains SB3 and GN101. All strains share 16S rRNA sequence identities of 98 to 99% to Hbt. salinarum R1, but possess distinct plasmid populations different from the three wild type strains (Ebert et al., 1984). Hbt. salinarum R1 and DSM 3754T _{show strong, Hbt. salinarum PHH4 and SB3 moderate adhesion to plastic}

surfaces, whereas no significant adhesion is observed for Hbt. salinarum NRC-1, PHH1, and GN101 (Fröls et al., 2012).

A genotyping analysis was applied to test for a correlation between the identified type IV pili systems pil-1 and pil-2, as well as the putative pilin genes and the adhesion capabilities of these seven halobacterial strains. Total DNA of the strains was hydrolyzed with AatII and blotted as described in 2.4.11. Southern hydridizations were done using probes specific for genes of the pil loci as well as for genes putatively encoding pilins.

Figure 29 Southern analyses investigating the occurrence of the pil-1and pil-2loci in different strains of

Hbt. salinarum. Total DNA samples were digested with AatII. A, Hbt. salinarum R1 pil-1locus with the central genes pilB1 and pilC1. AatII restriction sites and resulting DNA fragment sizes are indicated. Probe hybridization region is shaded. B, Hbt. salinarum R1 pil-2 locus with the central genes pilB2 and pilC2,

AatII restriction sites and resulting DNA fragment size as well as probe hybridization region (shaded). C, Restriction patterns obtained after hydrolysis of genomic DNA of different Halobacterium salinarum

The pilB1/C1 probe hybridized with DNA of all strains, but strain-specific variations were observed with regard to the restriction fragments detected (Figure 29D). Five of the seven strains tested (R1, NRC-1, PHH1, PHH4, and SB3) displayed probe hybridizations with two fragments of 4.1 kbp and 3.3 kbp, corresponding to the theoretical sizes calculated from the genome sequence of Hbt. salinarum R1 (Figure 29A). However, only one size of the hybridizing restriction fragment (3.3 kbp) was detected for Hbt. salinarum DSM 3754T _{and GN101, while}

the thick bands might also result from two similar-sized fragments.

The pilB2 probe yielded even more divergent patters. The probe was expected to label a single fragment of 4.3 kbp, based on the Hbt. salinarum R1 genetic sequence (Figure 29B). Such a fragment was detected in Hbt. salinarum R1, DSM 3754T _{and GN101 (Figure 29E). In contrast,} Hbt. salinarum strains PHH4 and SB3 contained a 7 kbp fragment, whereas regarding the strains PHH1 and NRC-1 the fragments hybridizing were larger than 10 kbp.

Figure 30 Southern analyses examining the presence of putative pilin encoding genes in different strains of Hbt. salinarum (DSM 3754, R1, NRC-1, PHH1, PHH4, SB3 and GN101). Total DNA samples were digested with AatII. Pilin gene-specific probes were employed. The respective gene/s tested is/are indicated below the hybridization patterns. A simultaneous detection of pilA2-4 was possible by use of pilA3-specific probes, since the three genes are part of a cluster. All probes were hybridized with the same blot after stripping, except for the pilA8 probes hybridized with a separate blot. Arrow heads mark restriction fragments expected with respect to strain Hbt. salinarum R1. Fragment sizes are indicated in Table 8.

Eight out of nine pilin probes tested gave hybridization signals in all Hbt. salinarum strains under investigation, suggesting that the putative pilin genes are conserved, with the exception of pilA7 (Figure 30). The pilA7 probe did not yield signals in the strains DMS 3754T _{and SB3,}

representing the best adhering and a weakly adhering strain of Halobacterium, respectively [Table 8; (Fröls et al., 2012)], while the signals in the other stains were similar to strain R1.

Although the other probes gave rise to signals in all strains, in some cases differing restriction fragments were detected. With regard to the pilA1 and pilA11 probes, only one of the other strains yielded the same signal as strain R1, i.e. PHH4 and SB3, respectively. Both strains have weak adhesion capabilities as demonstrated previously (Fröls et al., 2012). In both cases strain DSM 3754T_{, which displays the strongest adhesion of the halobacterial strains tested (Fröls et} al., 2012), resulted in unique hybridization signals. A unique restriction fragment of strain DSM 3754T _{was also detected using a pilA8 specific probe, while the other strains gave similar signals}

as strain R1. With regard to the pilA2-4, pilA5, pilA6 and pilA10 specific probes, similar restriction fragments were observed in all strains. Regarding the pilA9 probe, only the non- adherent strain GN101 (Fröls et al., 2012) displayed a unique fragment, while the other strains appeared similar.

Table 8 Adhesion properties of different Hbt. strains and detection of putatively T4P associated genes Hbt. salinarum strains

DSM

3754T R1 NRC-1 PHH1 PHH4 SB3 GN101

Adhesion strong strong no no weak weak no

Targe t ge ne s pilB1-C1 (+) 3.3 4.1 + + + + (+) D et ect ed f rag me nt si ze s [kb p ] pilB2 + 4.3 (+) (+) (+) (+) + pilA1 (+)U _{2.1 (+) (+) + (+) (+)} pilA2-4 + 1.8 2.0 + + + + + pilA5 + 2.2 + + + + + pilA6 + 3.6 + + + + + pilA7 – 4.0 + + + – + pilA8 (+)U _{1.3 + + + + +} pilA9 + 0.6 2.6 + + + + (+) pilA10 + 3.9 + + + + + pilA11 (+)U _{4.7 (+) (+) (+) + (+)}

*Information on adhesion properties obtained from Fröls et al. (2012). +, similar restriction fragment size detected as for strain R1

(+), hybridization signal of different size compared to strain R1 U_{, unique restriction fragment in a certain strain}

–, no hybridization signal

adhesion ability, while the 10 kbp fragments of strains NRC-1 and PHH1 coincide with their inability to adhere. Regarding the putative pilin genes, differential restriction patterns were observed upon hybridization with the pilA1, pilA8, pilA9 and pilA11 probes, while the other probes resulted in similar signals in all strains tested. Only the pilA7 probe did not show hybridizations in all strains.

4.3. Discussion

4.3.1. Hbt. salinarum R1 possesses two type IV pili systems besides the archaella operon