R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors ...like heavy metals on biofilms was still unknown. Here, we report on the first insights into
biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on
and imply a metal-specific protective response of cells in biofilms.
Archaeal biofilms: the great unexplored Orell, Alvaro; Fröls, Sabrina; Albers, Sonja-Verena
Annual review of microbiology,
01/2013, Letnik:
67
Journal Article
Recenzirano
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.
Biofilm formation by haloarchaea Fröls, Sabrina; Dyall-Smith, Mike; Pfeifer, Felicitas
Environmental microbiology,
December 2012, Letnik:
14, Številka:
12
Journal Article
Recenzirano
Summary
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.
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.
Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against ...various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained Km and kcat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s(-1), respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (Ki of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn2+-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.
Summary
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.
Summary
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.
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.
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