Sabrina Fröls

UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV‐induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre‐pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV‐treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV‐inducible pili operon of Sulfolobus. Overexpression of the pre‐pilins, Ups‐A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV‐dose‐dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double‐strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Response of the Hyperthermophilic Archaeon Sulfolobus solfataricus to UV Damage

In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV‐inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species‐specific recognition process and that UV‐induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self‐aggregate, but were partners in mating experiments with wild‐type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV‐inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.

Biofilm formation by haloarchaea

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value  < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

Archaeal biofilms: widespread and complex

Biofilms or multicellular structures become accepted as the dominant microbial lifestyle in Nature, but in the past they were only studied extensively in bacteria. Investigations on archaeal monospecies cultures have shown that many archaeal species are able to adhere on biotic and abiotic surfaces and form complex biofilm structures. Biofilm-forming archaea were identified in a broad range of extreme and moderate environments. Natural biofilms observed are mostly mixed communities composed of archaeal and bacterial species of various abundances. The physiological functions of the archaea identified in such mixed communities suggest a significant impact on the biochemical cycles maintaining the flow and recycling of the nutrients on earth. Therefore it is of high interest to investigate the characteristics and mechanisms underlying the archaeal biofilm formation. In the present review, I summarize and discuss the present investigations of biofilm-forming archaeal species, i.e. their diverse biofilm architectures in monospecies or mixed communities, the identified EPSs (extracellular polymeric substances), archaeal structures mediating surface adhesion or cell-cell connections, and the response to physical and chemical stressors implying that archaeal biofilm formation is an adaptive reaction to changing environmental conditions. A first insight into the molecular differentiation of cells within archaeal biofilms is given.

Archaeal Biofilms: The Great Unexplored

Biofilms are currently viewed as the most common form in which microorganisms exist in nature. Bacterial biofilms play important roles in disease and industrial applications, and they have been studied in great detail. Although it is well accepted that archaea are not only the extremists they were thought to be as they occupy nearly every habitat where also bacteria are found, it is surprising how little molecular details are known about archaeal biofilm formation. Therefore, we aim to highlight the available information and indicate open questions in this field.

Reactions to UV damage in the model archaeon Sulfolobus solfataricus

Mechanisms involved in DNA repair and genome maintenance are essential for all organisms on Earth and have been studied intensively in bacteria and eukaryotes. Their analysis in extremely thermophilic archaea offers the opportunity to discover strategies for maintaining genome integrity of the relatively little explored third domain of life, thereby shedding light on the diversity and evolution of these central and important systems. These studies might also reveal special adaptations that are essential for life at high temperature. A number of investigations of the hyperthermophilic and acidophilic crenarchaeote Sulfolobus solfataricus have been performed in recent years. Mostly, the reactions to DNA damage caused by UV light have been analysed. Whole-genome transcriptomics have demonstrated that a UV-specific response in S. solfataricus does not involve the transcriptional induction of DNA-repair genes and it is therefore different from the well-known SOS response in bacteria. Nevertheless, the UV response in S. solfataricus is impressively complex and involves many different levels of action, some of which have been elucidated and shed light on novel strategies for DNA repair, while others involve proteins of unknown function whose actions in the cell remain to be elucidated. The present review summarizes and discusses recent investigations on the UV response of S. solfataricus on both the molecular biological and the cellular levels.

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

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.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

Shedding light on biofilm formation of Halobacterium salinarum R1 by SWATH-LC/MS/MS analysis of planktonic and sessile cells.

Early and mature biofilm formation in the extremely halophilic euryarchaeon Halobacterium salinarum strain R1 was characterized by SWATH‐LC/MS/MS. Using a simple surfactant‐assisted protein solubilization protocol and one‐dimensional ultra‐high performance nanoflow chromatography on the front end, 63.2 and 58.6% of the predicted H. salinarum R1 proteome could be detected and quantified, respectively. Analysis of biophysical protein properties, functional analysis and pathway mapping indicated comprehensive characterization of the proteome. Sixty point eight percent of the quantified proteins (or 34.5% of the predicted proteome) exhibited significant abundance changes between planktonic and sessile states, demonstrating that haloarchaeal biofilm formation represents a profound “lifestyle change” on the molecular level. Our results and analysis constitute the first comprehensive study to track molecular changes from planktonic cultures to initial and mature archaeal biofilms on the proteome level. Data are available via ProteomeXchange, identifier PXD003667. Proteins exemplifying different protein expression level profiles were selected, and their corresponding gene transcripts targeted by qRT‐PCR to test the feasibility of establishing rapid PCR‐based assays for archaeal biofilm formation.