Outside the Unusual Cell Wall of the Hyperthermophilic Archaeon Aeropyrum pernix K1

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.The archaea are a unique group of organisms that share properties with both the eukarya and bacteria. For a long time, archaeal life was considered to be limited to extreme environments such as high temperature, alkaline and acidic hot springs, anaerobic sediments, and highly saline environments. In the last decade, by the use of the archaeal 16 S rRNA gene as a molecular marker in microbial surveys (1), numerous mesophilic species have also been detected (2). Archaea have been found frequently and sometimes closely associated with bacterial and eukaryotic host cells, including humans. One of the most intriguing aspects of archaea is their unusual barrier between the inner cell material and the cellular environment, i.e. their cell membrane. Biosynthesis of archaeal cell wall has been a subject of interest for a long time. Most of the archaeal species characterized so far have a single chemically distinct cell membrane, which differs considerably from their eukaryotic and bacterial counterparts (3). The ether-type polar lipid surface is covered by a surface layer (S-layer)¹ composed of glycoproteins crystallized in regular two-dimensional lattices with hexagonal or tetragonal symmetry (4, 5). The structural characterization of the S-layer (6, 7) and S-layer-embedded archaeal cellular appendices such as flagella (8), pili, and hami (7, 9) associated with a diverse arsenal of cellular functions like motility, cell-cell communication, signaling, adherence, and nutrient uptake, has been the subject of an increasingly significant number of studies. Protein secretion mechanisms through this unusual cell membrane have been mainly addressed by way of comparative genomics studies (10–12) and by genomic identification and characterization of signal peptidases (13, 14). Archaeal extracellular and cell membrane proteins have been predicted because of the presence of a tripartite N-terminal signal motif essential for protein secretion and subsequently cleaved by signal peptidases from the protein (11, 14, 15). In archaea three different signal peptidases have been identified and characterized so far (13): signal peptidase I is responsible for the cleavage of secretory signal peptides from the majority of secreted proteins, class III signal peptidase is responsible for processing signal peptides from preflagellins and some sugar-binding proteins (11), and signal peptide peptidase is responsible for the hydrolysis of signal peptides following protein secretion. No signal peptidase II homolog in archaea has been described to date. Four distinct pathways have been proposed for archaeal protein export: the main “Sec” system, the twin arginine translocation or “Tat” pathway (12), the ATP-binding cassette (ABC) transport system (16), and the type IV prepilin-like pathway (11). Moreover, proteins without signal peptides could also be secreted by using nonspecific and/or currently unknown mechanisms. Despite the similarities in protein translocation mechanisms between the three domains of life, genome analyses also shed light on unique archaeal characteristics, suggesting that our current knowledge regarding secreted proteins and secretion mechanisms in archaea remains limited (10). It is apparent that the lack of experimental data at the proteome level has become the bottleneck for the further understanding of the existence of novel secretion mechanisms in archaea (15).To date, the genome sequences of eight hyperthermophiles, including the crenarchaeon Aeropyrum pernix K1, have been determined. A. pernix K1, isolated from a coastal solfataric thermal vent on the Kodakara-Jima Island in Japan (17), is the first reported obligate aerobic and neutrophilic hyperthermophilic archaeon with an optimal growth temperature between 90 and 95 °C. The spherical shaped cells of A. pernix are ∼1 μm in diameter, lack a rigid cell wall, and are covered by an S-layer with hexagonal symmetry. A. pernix, like other extreme thermophiles and acidophiles, possesses a particularly thick cell membrane that acts as a protective barrier, conferring to it the ability to function in the extreme environment in which it thrives. The lipids of A. pernix are different from those of anaerobic sulfur-dependent hyperthermophiles; they lack tetraether lipids and the direct linkage of inositol and sugar moieties (18). A. pernix K1 contains a 1.6-Mbp chromosome that has been sequenced; it comprises 1700 annotated genes. By using different proteomics approaches, the proteome of A. pernix K1 has recently been analyzed, leading to the identification of 704 proteins (41% of total ORFs) (19). In this work we performed proteomics analysis of the cell surface and extracellular protein fractions purified from A. pernix K1 to define proteins targeted to the cell secretome. We also analyzed the complete predicted proteome of A. pernix K1 by in silico signal peptide and cellular localization prediction tools and compared the experimentally obtained data set with the predicted secretome.