Shedding light on adhesion and biofilms of Halobacterium salinarum R1

Biofilms, i.e. multicellular microbial communities, are widely accepted as the predominating mode of prokaryotes living in nature. However, knowledge about this lifestyle is still limited, especially in Archaea. The present work focuses on the formation of archaeal biofilms by the extremely halophilic archaeon Halobacterium salinarum R1. Surface adhesion of Hbt. salinarum R1 was monitored by phase contrast microscopy and quantified in a fluorescence-based adhesion assay, and demonstrated that abiotic surfaces were successively colonized by the cells. The formation of complex three-dimensional cell clusters with tower-like structures up to 25 µm in height was observed within 15 days by scanning electron microscopy and confocal laser scanning microscopy. Extracellular polymeric substances, i.e. a complex biofilm matrix containing extracellular DNA and glycosidic residues, was detected using suitable molecular probes, as well as a high viability of the biofilm cells. The sequence of events observed during the biofilm formation consisted of adhesion, accumulation and maturation. Adherent cells contained different types of cell surface structures, since filaments with two predominant diameters (7-8 and 10 nm) were observed. One of the diameters belongs to the archaellum, whereas the smaller one belongs to pili involved in adhesion. The Hbt. salinarum R1 genome was searched for genes potentially associated with the synthesis of cell surface structures by bioinformatical analyses. Two gene loci, pil-1 and pil-2, putatively encoding type IV pilus-like structures were identified. It was demonstrated by RT-PCR that both loci were transciptionally active and cotranscribed. Moreover, qRT-PCR yielded 5.2- and 8.5-fold induction of the respective ATPase genes, pilB1 and pilB2, in adherent cells compared to planktonic cells. Deletion of the archaella ATPase gene, flaI, resulted in cells lacking the 10 nm filaments. These cells were non-motile but still showed the 7-8 nm appendages and strong adhesion. An additional deletion of pilB1 in a ΔflaI/ΔpilB1 mutant severely impaired the ability of the cells to adhere, which was reduced to 20% compared to the parental strain. In contrast, an additional deletion of pilB2 did not have further effects on adhesion. A search for genes encoding the filament subunits, i.e. pilins, yielded more than 30 candidates. Transcriptional analyses of the most likely candidates demonstrated differential expression of the genes in planktonic and adherent samples, with the genes pilA5, pilA6 and pilA7 showing 2.5- to 7.1-fold induction in initial biofilms. A proteome analysis of the biofilm formation was performed investigating planktonic as well as initial and mature biofilm cells of Hbt. salinarum R1. A molecular differentiation of the protein pattern was already observed by SDS-PAGE in samples derived from biofilms after one day compared to planktonic cells. Employing label-free mass spectrometric SWATH-LC/MS/MS analysis a high coverage of the predicted proteome was achieved, reflected by 1629 different proteins identified and 1464 proteins quantified (63.2% and 56.8% of the total proteome, respectively). A relative quantification was performed, showing between 55 and 245 proteins strongly altered (> 2-fold) when two of the cellular states were compared. 882 proteins showed statistically significant abundance changes, correspoding to 60.8% of the quantified proteins and 34.2% of the total proteome, respectivly, reflecting the high diversity of the processes affected. The relative changes detected ranged between 195-fold increase of an uncharacterized glutamine-rich alkaline protein (OE3542R) and 22.8-fold decrease of ribonucleoside-diphosphate reductase subunit beta (NrdB1). The most striking effects were observed with proteins involved in energy conversion, as well as proteins acting in nucleotide-, amino acid- and lipid metabolism. In addition, proteins associated with protein biosynthesis and cellular processes like cell motility and signal transduction were strongly affected. The proteomic data of selected proteins was validated by qRT-PCR transcriptional analyses. This work represents the first comprehensive description of haloarchaeal biofilm formation using the example of Hbt. salinarum R1