Novel pili-like surface structures of Halobacterium salinarum strain R1 are crucial for surface adhesion

Abstract

It was recently shown that haloarchaeal strains of different genera are able to adhere to surfaces and form surface-attached biofilms. However, the surface structures mediating the adhesion were still unknown. We have identified a novel surface structure with Halobacterium salinarum strain R1, crucial for surface adhesion. Electron microscopic studies of surface-attached cells frequently showed pili-like surface structures of two different diameters that were irregularly distributed on the surface. The thinner filaments, 7-8 nm in diameter, represented a so far unobserved novel pili-like structure. Examination of the Hbt. salinarum R1 genome identified two putative gene loci (pil-1 and pil-2) encoding type IV pilus biogenesis complexes besides the archaellum encoding fla gene locus. Both pil-1 and pil-2 were expressed as transcriptional units, and the transcriptional start of pil-1 was identified. In silico analyses revealed that the pil-1 locus is present with other euryarchaeal genomes whereas the pil-2 is restricted to haloarchaea. Comparative real time qRT-PCR studies indicated that the general transcriptional activity was reduced in adherent vs. planktonic cells. In contrast, the transcription of pilB1 and pilB2, encoding putative type IV pilus assembly ATPases, was induced in comparison to the archaella assembly/motor ATPase (flaI) and the ferredoxin gene. Mutant strains were constructed that incurred a flaI deletion or flaI/pilB1 gene deletions. The absence of flaI caused the loss of the archaella while the additional absence of pilB1 led to loss of the novel pili-like surface structures. The ΔflaI/ΔpilB1 double mutants showed a 10-fold reduction in surface adhesion compared to the parental strain. Since surface adhesion was not reduced with the non-archaellated ΔflaI mutants, the pil-1 filaments have a distinct function in the adhesion process.

Keywords: Halobacterium salinarum; archaeal type IV pili; archaellum; deletion mutant; haloarchaea; surface adhesion.

Figure 1

TEM analyses of Hbt. salinarum R1 attached to surfaces. (A) Surface attached cells grown for 10 d at 42°C on carbon coated gold grids. (B,C) Pili-like surface structures observed on the carbon surface. (D,E) Determination of different diameters of pili-like structures. Arrow heads, wide diameters; arrows, thinner structures. (F) Frequency distribution observed with different diameters determined for 100 surface structures.

Figure 2

Genomic regions and transcriptional analyses of the pil-1 and pil-2 loci. (A) Top: pil-1 locus with genes encoding the putative type IV pili assembly ATPase (pilB1) and putative transmembrane protein (pilC1) marked in black. Bottom: RT-PCR to determine a putative co-transcription using oligonucleotides amplifying fragments across the intergenic regions (brackets numbered 1 to 5 above the gel correspond to fragments 1 to 5 in the gene map; dashed lines, no co-transcription detected; full lines, co-transcription detected). For each pair of adjacent genes the three lanes in the gel represent (a) PCR product using Hbt. salinarum R1 genomic DNA as template to validate the amplicon size and oligonucleotides specificity; (b) PCR product with RNA of planktonic Hbt. salinarum R1 cells without reverse transcription; (c) RT-PCR product. (B) Upstream and 5′ nucleotide sequence of pilB1 (OE2215R, shown in grey) and the AUG translation start codon predicted for OE2215R is marked by a dashed box. The transcription start site determined by primer extension is labeled +1. The alternative GUG translation start codon is boxed. (C) Top: pil-2 locus with genes encoding the putative type IV pili assembly ATPase (pilB2) and putative transmembrane protein (pilC2) marked in black. Putative prepilin encoding genes are shown in grey. Bottom: RT-PCR experiment investigating co-transcription of the pil-2 genes similarly to pil-1 as explained in 2A. Brackets numbered 1 to 8 in the gel correspond to fragments 1 to 8 in the gene map.

Figure 3

Comparative qRT-PCR analyses of planktonic and surface attached cells. (A) Investigation of two representative housekeeping genes encoding RNA polymerase subunit B' (rpoB1) and the translation elongation factor 2 (aef2). Relative expression was normalized to external standard bgaH RNA. (B) Relative transcriptional quantification of the assembly ATPase encoding genes of the archaellum (flaI) and the type IV pilus biogenesis complexes 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.

Figure 4

Motility assay of Hbt. salinarum R1, ΔflaI and ΔflaI/ΔpilB1 mutant strains. Swimming motility of Hbt. salinarum R1 as well as ΔflaI and ΔflaI/ΔpilB1 mutant strains was analyzed in semi solid plates with 0.3% (w/v) agar 3 days after cultivation at 42°C. The scale bar on the right is 1 cm.

Figure 5

Phenotypic characterization of the Hbt. salinarum R1, ΔflaI and ΔflaI/ΔpilB1 mutant strains. (A) Transmission electron micrographs of surface attached cells on carbon coated gold grids after 10 days of cultivation at 42°C. Pili-like surface structures observed with ΔflaI are labeled with arrows. (B) Frequency distribution of 100 filaments diameters in nm found with the ΔflaI mutant. (C) Light micrographs of Hbt. salinarum R1 and mutant strains attached to a glass surface after 10 days of cultivation at 42°C. Cells not attached to the surface were removed by stringent washing. Scale bars are 10 μm.