No abstract

Takamura et al. show that most lung CD8+ TRM cells are not maintained in the inducible bronchus-associated lymphoid tissue (iBALT) but are maintained in specific niches created at the site of tissue regeneration, which are termed as repair-associated memory depots (RAMDs).

The membrane-embedded rotor in the F 0 sector of protontranslocating ATP synthases is formed from hairpin-like c-subunits that are protonated and deprotonated during energization of ATP synthesis. This study focuses on two c-subunit motifs that are unique to synthases of extremely alkaliphilic Bacillus species. One motif is the AXAXAXA sequence found in the N-terminal helix-1 instead of the GXGXGXG of non-alkaliphiles. Quadruple A3 G chromosomal mutants of alkaliphilic Bacillus pseudofirmus OF4 retain 50% of the wild-type hydrolytic activity (ATPase) but <18% of the ATP synthase capacity at high pH. Consistent with a structural impact of the four alanine replacements, the mutant ATPase activity showed enhanced inhibition by dicyclohexylcarbodiimide, which blocks the helix-2 carboxylate. Single, double, or triple A3 G mutants exhibited more modest defects, as monitored by malate growth. The key carboxylate is in the second motif, which is P 51 XXE 54 XXP in extreme alkaliphiles instead of the (A/G)XX(E/ D)XXP found elsewhere. Mutation of Pro 51 to alanine had been shown to severely reduce malate growth and ATP synthesis at high pH. Here, two Pro 51 to glycine mutants of different severities retained ATP synthase capacity but exhibited growth deficits and proton leakiness. A Glu 54 to Asp 54 change increased proton leakiness and reduced malate growth 79 -90%. The Pro 51 and the Glu 54 mutants were both more dicyclohexylcarbodiimide-sensitive than wild type. The results highlight the requirement for c-subunit adaptations to achieve alkaliphile ATP synthesis with minimal cytoplasmic proton loss and suggest partial suppression of some mutations by changes outside the atp operon.

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+-instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.

Alkaliphiles are microorganisms that can grow in alkaline environments, i.e. pH >9.0. Their enzymes, especially extracellular enzymes, are able to function in their catalytic activities under high alkaline pH values because of their stability under these conditions. Proteases, protein degrading enzymes, are one of the most produced enzymes in industry. Among proteases, alkaline proteases, which are added to some detergents, are the most produced. Other alkaline enzymes, e.g. alkaline cellulases, alkaline amylases, and alkaline lipases, are also adjuncts to detergents for improving cleaning efficiency. Alkaline enzymes often show activities in a broad pH range, thermostability, and tolerance to oxidants compared to neutral enzymes. Alkaliphilic Bacillus species are the most characterized organisms among alkaliphiles. They produce so many extracellular alkaline-adapted enzymes that they are often good sources for industrial enzymes. As a patent strain, the whole genome sequence of alkaliphilic Bacillus halodurans C-125 has been sequenced for the first time. In addition, an increasing number of whole genomic sequences and structural analyses of proteins in alkaliphiles, development of genetic engineering techniques and physiological analyses will reveal the alkaline adaptation mechanisms of alkaliphilic Bacillus species and the structural basis of their enzymatic functions. This information opens up the possibility of new applications. In this paper we describe, first, the physiologies of environmental adaptations, and then the applications of enzymes and microorganisms themselves in alkaliphilic Bacillus species.