Makoto Fujisawa

Industrial applications of alkaliphiles and their enzymes – past, present and future

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.

F1F0-ATP synthases of alkaliphilic bacteria: lessons from their adaptations.

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 a 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.

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Properties of four two-component bacterial transport systems of the cation/proton antiporter-2 (CPA2) family led to suggestions that this CPA2 subset may use a channel rather than an antiport mechanism [see Booth IR, Edwards MD, Gunasekera B, Li C, Miller S (2005) in Bacterial Ion Channels, eds Kubalski A, Martinac B (Am Soc Microbiol, Washington, DC), pp 21–40]. The transporter subset includes the intensively studied glutathione-gated K+ efflux systems from Escherichia coli, KefGB, and KefFC. KefG and KefF are ancillary proteins. They are peripheral membrane proteins that are encoded in operons with the respective transporter proteins, KefB and KefC, and are required for optimal efflux activity. The other two-component CPA2 transporters of the subset are AmhMT, an NH4+ (K+) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K+ efflux system from Bacillus subtilis. Here a K+/H+ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent Km for K+ was in the low mM range. The peripheral protein was required for YhaU- and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb+≈K+>Li+>Na+. Glutathione significantly inhibited KefFC-mediated K+/H+ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H+ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH4+, the results also imply that AmhMT catalyzes NH4+/H+ antiport, which would prevent net cytoplasmic H+ loss during NH4+ efflux.

Characterization of the Functionally Critical AXAXAXA and PXXEXXP Motifs of the ATP Synthase c-Subunit from an Alkaliphilic Bacillus

The membrane-embedded rotor in the F0 sector of proton-translocating 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 A→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 A→G mutants exhibited more modest defects, as monitored by malate growth. The key carboxylate is in the second motif, which is P51XXE54XXP in extreme alkaliphiles instead of the (A/G)XX(E/D)XXP found elsewhere. Mutation of Pro51 to alanine had been shown to severely reduce malate growth and ATP synthesis at high pH. Here, two Pro51 to glycine mutants of different severities retained ATP synthase capacity but exhibited growth deficits and proton leakiness. A Glu54 to Asp54 change increased proton leakiness and reduced malate growth 79-90%. The Pro51 and the Glu54 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.

An Intergenic Stem-Loop Mutation in the Bacillus subtilis ccpA-motPS Operon Increases motPS Transcription and the MotPS Contribution to Motility

A stem-loop mutation between ccpA and motP in the Bacillus subtilis ccpA-motPS operon increased motPS transcription and membrane-associated MotPS levels, motility, and number of flagella/cell when MotPS is the sole stator and the MotPS contribution to motility at high pH, Na+, and viscosity when MotAB is also present.

Modulation of the K+ efflux activity of Bacillus subtilis YhaU by YhaT and the C-terminal region of YhaS

The cation/proton antiporter 2 (CPA2) family is a large family of cation transporters and putative channel proteins that are found in bacteria, archaea as well as eukaryotes. Consistent with a K+ efflux capacity that is found in several other CPA2 proteins, it is shown here that the YhaU protein of Bacillus subtilis greatly increased the concentration of K+ required for growth of a K+ uptake-defective mutant of Escherichia coli. No YhaU-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles. Two genes, yhaS and yhaT, are located upstream of yhaU and form an apparent operon with it. The YhaS protein has no reported homologues while the YhaT protein has sequence similarity to a sub-domain of KTN proteins that are associated with potassium-translocating channels and transporters. YhaT and the C-terminal region of YhaS were shown to modulate the K+ transport capacities of YhaU in complementation experiments. Expression studies, conducted by monitoring the beta-galactosidase levels in pMutin-disrupted mutants of the yhaU locus, indicated that yhaU is strongly induced by alkaline pH- plus salt-induced stress and that there are additional sodium-specific responses of yhaS and yhaT.

The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant MUTATIONS IN THE CRITICAL ALKALIPHILE-SPECIFIC RESIDUE LYS-180 AND OTHER RESIDUES THAT SUPPORT ALKALIPHILE OXIDATIVE PHOSPHORYLATION

A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and β-subunit levels at least as high as wild-type. Purified mutant F1F0-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395–17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness.