Macromolecular Fingerprinting of Sulfolobus Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis

Microorganisms in nature often live in surfaceassociated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development

Cell Surface Structures of Archaea

Prokaryotes possess various kinds of cell surface organelles serving versatile biological roles depending on the environmental niche of the organism. The formation of these structures involves fascinating machineries, as not only do the protein components need to travel across the cytoplasmic membrane like all secreted proteins, but they also need to do so in a precisely coordinated manner for proper assembly. Most commonly found on the surface of bacteria are flagella used for swimming (47); the type III secretion injectisome (needle structure) (21), which is used to deliver effector molecules from pathogenic organisms into host cells; and a wide variety of thinner organelles that fall under the broad designation of pili (13, 33, 58, 64, 69, 78). Different classes of these structures (type I pili, type IV pili, sex pili, etc.) which differ significantly in their structure, assembly, and function have been identified. Their many roles include adhesion, twitching (or surface) motility, and delivery of DNA and toxins, as well as functioning as electrically conductive “nanowires.” Other, less commonly studied appendages have also been reported, such as spinae (9)

Cell Surface Structures of Archaea

Prokaryotes possess various kinds of cell surface organelles serving versatile biological roles depending on the environmental niche of the organism. The formation of these structures involves fascinating machineries, as not only do the protein components need to travel across the cytoplasmic membrane like all secreted proteins, but they also need to do so in a precisely coordinated manner for proper assembly. Most commonly found on the surface of bacteria are flagella used for swimming (47); the type III secretion injectisome (needle structure) (21), which is used to deliver effector molecules from pathogenic organisms into host cells; and a wide variety of thinner organelles that fall under the broad designation of pili (13, 33, 58, 64, 69, 78). Different classes of these structures (type I pili, type IV pili, sex pili, etc.) which differ significantly in their structure, assembly, and function have been identified. Their many roles include adhesion, twitching (or surface) motility, and delivery of DNA and toxins, as well as functioning as electrically conductive “nanowires.” Other, less commonly studied appendages have also been reported, such as spinae (9)

A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins.

Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein

Trapped in action: direct visualization of DNA methyltransferase activity in living cells

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2′-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells

A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells

Genetic high-throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid (F2H) assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the F2H assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real-time

Targeting and tracing antigens in live cells with fluorescent nanobodies

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion