Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis

Abstract

Natronomonas pharaonis is an extremely haloalkaliphilic archaeon that was isolated from salt-saturated lakes of pH 11. We sequenced its 2.6-Mb GC-rich chromosome and two plasmids (131 and 23 kb). Genome analysis suggests that it is adapted to cope with severe ammonia and heavy metal deficiencies that arise at high pH values. A high degree of nutritional self-sufficiency was predicted and confirmed by growth in a minimal medium containing leucine but no other amino acids or vitamins. Genes for a complex III analog of the respiratory chain could not be identified in the N. pharaonis genome, but respiration and oxidative phosphorylation were experimentally proven. These studies identified protons as coupling ion between respiratory chain and ATP synthase, in contrast to other alkaliphiles using sodium instead. Secretome analysis predicts many extracellular proteins with alkaline-resistant lipid anchors, which are predominantly exported through the twin-arginine pathway. In addition, a variety of glycosylated cell surface proteins probably form a protective complex cell envelope. N. pharaonis is fully equipped with archaeal signal transduction and motility genes. Several receptors/transducers signaling to the flagellar motor display novel domain architectures. Clusters of signal transduction genes are rearranged in haloarchaeal genomes, whereas those involved in information processing or energy metabolism show a highly conserved gene order.

Figure 1.

Schematic representation of the nitrogen metabolism of N. pharaonis. The figure illustrates proposed transport (green arrows) and metabolic processes (blue arrows) for nitrogen compounds and the associated gene clusters (nark–narB–nirA, urtABCDE, ureBACGDEF). Ammonia can be supplied by (1) direct uptake, (2) uptake and reduction of nitrate () via nitrite (), and (3) uptake and hydrolysis of urea. It is utilized by a two-step reductive conversion of 2-oxoglutarate (2-OG) to glutamate (Glu) involving ferredoxin (fdx). The two glnA paralogs, gltB, and amtB were found distributed over the genome.

Figure 2.

Genomic and experimental studies of the N. pharaonis electron transport chain. (A) The electron transport chain profile for several respiratory archaea displays subunits (squares) of respiratory complexes (boxes I–IV). These subunits are often encoded adjacently in the archaeal genomes (straight connections) and can be fused to each other (curved connections). The proposed electron flow between respiratory complexes is indicated by arrows. Complexes, which have been characterized experimentally, are indicated in blue, and protein-sequenced subunits of isolated complexes are yellow. The profile for Sulfolobus acidocaldarius (Sa) was established by complementation with genetic data from the completely sequenced Sulfolobus solfataricus (Ss) (asterisks). Menaquinone (MQ), caldariellaquinone (CQ), halocyanin (Hcy), and sulfocyanin (Scy) are shown or predicted to function as mobile carriers in archaeal respiratory chains. Homologs to complex I subunits have been found in archaeal genomes, but genes encoding the NADH acceptor module are missing. A functional NADH-dehydrogenating complex I has been experimentally excluded for three species (red). Instead, it is replaced by NADH dehydrogenase type II, which is not capable of proton translocation. (B,C) Oxidative and photo-phosphorylation processes in N. pharaonis cells were investigated through measurements of ATP levels (upper curves, mean values with error bars from triplicates) and extracellular pH (lower curves, continuous recording at a rate of 10/sec) in the (B) absence or (C) presence of the protonophore CCCP (0.2 mM). The effects (ON: above curve arrow, OFF: below curve arrow) of light (hν, λ > 515 nm, 32 mW/cm²) and aeration (O₂) were determined. All experiments were performed at pH 8.1. Vertical scaling bars indicate ATP level and amount of proton uptake, whereas horizontal scaling bars indicate a 10-min time interval.

Figure 3.

Schematic representation of protein secretion, anchoring, and glycosylation in N. pharaonis. (A) Substrates of the Tat, Sec and flagellin-specific protein translocation systems (blue boxes) are cleaved by signal peptidases (flash signs), and partly remain C- or N-terminally anchored to the cell membrane. Secreted proteins are cleaved by signal peptidase type I (blue), whereas lipobox-containing proteins are cleaved by signal peptidase type II (orange) and N-terminally attached to a lipid anchor (orange box). Lipoproteins are frequently transported via the Tat pathway (substrate numbers indicated in light-blue arrows). For three lipobox-containing proteins, the export pathway remains as yet unassigned. Furthermore, six proteins are likely to be modified by a C-terminally attached lipid anchor (yellow box). After cleavage by membrane-bound preflagellin peptidase (green), the substrates of the flagellin-specific export pathway reveal an N-terminal hydrophobic stretch possibly involved in membrane retention. (B) Signal sequence and peptide repeat modules (indicated by colored boxes) for a representative gene cluster (white arrows) are presented diagrammatically in models of the cell surface proteins. A Thr-rich tetrapeptide repeat (red box in genes, red line in proteins), likely to be O-glycosylated, occurs in several cell surface proteins adjacent to the C-terminal or N-terminal lipid anchor. An Asn-Gln dipeptide repeat (gray box, oval) follows directly after the lipobox-containing Tat-related signal sequence (orange box) of several membrane components. Other indicated features are Sec-related signal sequences (blue box in genes) and N-glycosylation sites (red hexagons in proteins).

Figure 4.

Domain architecture and gene context of several transducers from Natronomas and Halobacterium. (A) Two groups of transducers (upper boxes) with distinct domain architectures and their adjacent genes (arrows and lower boxes) from Natronomonas (blue dots) and Halobacterium (red dots) are schematically represented. Transducers with a long extracellular domain between their two transmembrane domains (box TM2ED) are frequently involved in chemotaxis, and are cotranscribed with the genes for periplasmic substrate-binding proteins (box SBP). Several of the transducers with a short loop between their two transmembrane domains (short-loop transducers, box TM2SL) are cotranscribed with retinal-containing photoreceptors (box RP) or distant homologs thereof. Experimental environmental response data are indicated by colored arrows (green: chemotaxis [Kokoeva et al. 2002]; blue: blue-light phototaxis [Seidel et al. 1995; Zhang et al. 1996]; orange: orange-light phototaxis [Yao and Spudich 1992]). The existence of orthologous gene pairs in Haloarcula is indicated by asterisks. (B) The interaction between a short-loop transducer (TM2SL) and a retinal protein (RP) occurs within the membrane. The interaction between a chemotactic extracellular-domain transducer (TM2ED) with a lipid-anchored (yellow box) periplasmic substrate-binding protein (SBP) may occur outside of the membrane.