Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea. IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.
Motility is a central feature of many microorganisms and provides an efficient strategy to respond to environmental changes. Bacteria and archaea have developed fundamentally different rotary motors enabling their motility, termed flagellum and archaellum, respectively. Bacterial motility along chemical gradients, called chemotaxis, critically relies on the response regulator CheY, which, when phosphorylated, inverses the rotational direction of the flagellum via a switch complex at the base of the motor. The structural difference between archaellum and flagellum and the presence of functional CheY in archaea raises the question of how the CheY protein changed to allow communication with the archaeal motility machinery. Here we show that archaeal CheY shares the overall structure and mechanism of magnesium-dependent phosphorylation with its bacterial counterpart. However, bacterial and archaeal CheY differ in the electrostatic potential of the helix α4. The helix α4 is important in bacteria for interaction with the flagellar switch complex, a structure that is absent in archaea. We demonstrated that phosphorylation-dependent activation, and conserved residues in the archaeal CheY helix α4, are important for interaction with the archaeal-specific adaptor protein CheF. This forms a bridge between the chemotaxis system and the archaeal motility machinery. Conclusively, archaeal CheY proteins conserved the central mechanistic features between bacteria and archaea, but differ in the helix α4 to allow binding to an archaellum-specific interaction partner.
Bacteria and archaea employ rotating filamentous structures to swim through liquid environments. Although they are functionally similar, there is no structural similarity between the filaments from bacteria
In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.
A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0–10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The V max and K m values were 3.13 ± 0.47 U mg−1 and 2.94 ± 0.16 mg mL−1 for β-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed.
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