Influence of cell surface structures on crenarchaeal biofilm formation using a thermostable green fluorescent protein

Abstract

The thermoacidophilic crenarchaeote Sulfolobus acidocaldarius displays three distinct type IV pili-like structures on its surface: (i) the flagellum, (ii) the UV-induced pili and (iii) the adhesive pili. In bacteria, surface appendages play an important role in the spatial organization of cells from initial surface attachment to the development of mature community structures. To investigate the influence of the diverse set of type IV pili-like structures in S. acidocaldarius, single, double and triple mutants lacking the cell surface appendages were constructed and analysed for their behaviour in attachment assays and during biofilm formation. A heat stable green fluorescent protein was employed the first time in a hyperthermophilic archaeon. A codon adjusted eCGP123 was expressed to study mixed biofilms of different deletion mutants to understand the interplay of the surface structures during biofilm formation. During this process the deletion of the adhesive pili and UV-induced pili led to the most pronounced effects, either an increase in cell density or increased cluster formation respectively. However, all three cell surface appendages played a role in the colonization of surfaces and only the interplay of all three appendages leads to the observed wild-type biofilm phenotype.

Lrs14 transcriptional regulators influence biofilm formation and cell motility of Crenarchaea

“…Interestingly, this transcription profile resembled the one described for the S. acidocaldarius DflaJ strain (a non-archaellated strain) as it showed an increase of aapA and aapB transcript levels when grown as biofilms (Henche et al, 2011). The overexpression of aapA in the Dsaci0446 deletion strain might contribute its ability to form a more dense and stable biofilm because of the role of this adhesive appendage in both surface attachment and the establishment of cell-to-cell connections (Henche et al, 2011).…”

“…Indeed, the deletion of this regulator resulted in S. acidocaldarius cells that were noticeably impaired to build biofilms ( Figures 2 and 3). As CLSM analysis showed Dsaci1223 was impaired in surface colonization (B50% less cells on the surface), suggesting that the assembly of surface structures may be perturbed (Henche et al, 2011). However, none of the surface structure components, with the exception of upsAE, were found to be significantly altered in expression in biofilms formed by Dsaci1223 (Figure 4a).…”

“…As described by Henche et al (2011), after 3 days of growth reference strain MW001 showed a confluent dense biofilm, displaying an EPS pattern in which the ConA (mannose/glucose) signal was dominant (Figure 3). Biofilm communities formed by MW001 pyrEF þ showed no differences in comparison with MW001 (Supplementary Figure 5).…”

“…All three cell surface appendages played a role in surfaces colonization and the interplay of the three structures is important for the MW001 biofilm phenotype (Henche et al, 2011). Therefore, it was important to determine whether the deletion of saci1223, saci1242 or saci0446 led to an altered synthesis of any of these cell surface appendages and therefore to the observed Lrs14 deletion mutant biofilm phenotypes.…”

“…Therefore, it was important to determine whether the deletion of saci1223, saci1242 or saci0446 led to an altered synthesis of any of these cell surface appendages and therefore to the observed Lrs14 deletion mutant biofilm phenotypes. Using qRT-PCR, the expression levels of geneencoding components, which are essential for the assembly of each cell surface appendage (Henche et al, 2011), were determined in 3-day-old biofilm communities of Dsaci1223, Dsaci1242 and Dsaci0446, respectively. We determined that none of the tested cell surface appendage genes were differentially expressed in the Dsaci1242 deletion strain as their transcript levels were found to not be significantly altered (considering a threshold of X ± 2-fold changes; Figure 4a).…”