Abc 1630 Abc 1629

Fig. 4.14 pALK/ mrp/ sha Operon

in alkaliphilic B. halodurans,

B. pseudofirmus, B. clausii

Oceanobacillus iheyensis and B. subtilis subsp. sublilli 168.

Oceanobacillus iheyensis and B. subtilis subsp. sublilli 168.

Krulwich et al. 2001). The Na+/H+ antiporter activity is attributed to MrpA since the point mutation that leads to a non-alkaliphilic phenotype was in mrpA. The finding that a mutant in MrpC is also non-alkaliphilic (Seto et al. 1995) raises the possibility that additional mrp genes play critical roles even if MrpA contains the cation and proton translocation pathways. A requirement for multiple mrp genes in the extreme alkaliphiles is consistent with our inability to recover mutants of genetically accessible B. pseudo-firmus OF4 when attempts were made to disrupt any one of several mrp genes. The major Na +/H + antiporter of alkaliphilic Bacillus species may be required throughout their pH range. The Na+-specific monovalent cataion/proteon antiporter is necessary for the alkaliphile to maintain a cytoplasmic pH of 8.2 to 9.5 at external pH values from 10.5 to 11.2.

In neutrophilic B. subtilis, the Mrp system has a role in Na +-resistance and in both Na+-and K+-dependent alkaline pH homeostasis (Ito et al. 1999). Recently, Kosono et al. (2004) reported that a mrpA/shaA mutant of B. subtilis changes its use of the diverse ECF (extracytoplasmic function), oW and a-dependent transcription in transition phase this is consistent with complex stress reaction. In contrast to the central role of Mrp in pH homeostasis of alkaliphilic Bacillus species, Mrp is not the dominant antiporter in this process in B. subtilis, that role belongs to the multifunctional (tetracy-cline-divalent metal) + (Na + )(K + )/H+ antiporter Tet+ (Ito et al. 1999). On the other hand, the B. subtilis Mrp system plays a dominant role in Na resistance in this organisms, as indicated by the Na +-sensitive phenotypes of mrp/sha mutants. This result suggests that mrp is the essential gene of B. subtilis as examined on LB medium even though a mrp null strain is viable (Ito et al. 2000). As Swartz reviewed (2005), it will be of interest to compare the activity versus pH profile of alkaliphile and B. subtilis Mrp in many respects.

The other transport substrate for a Mrp system has been mentioned, i.e. a capacity for cholate efflux by the B. subtilis Mrp system (Ito et al. 1999; 2001). A mrp null strain of B. subtilis, from which the entire mrp operon is deleted, exhibits significantly reduced resistance to growth inhibition by the addition of cholate that is complemented by the introduction of the mrpF gene into the chromosomal amyE locus under the control of an IPTG-in-ducible promoter (Ito et al. 2000). Reduced cholate efflux was observed in starved whole cells of the mutant relative to the wild type. This defect is complemented significantly by the re-introduction of mrpF. Homology has been noted between MrpF and Na+-coupled bile transporters (Ito et al. 1999) and between MrpF and a region of voltage-gated Na+ channels (Mathiesen et al. 2003). However, no crucial experimental data for MrpF-mediated coupling between Na+ and cholate fluxes has been found (Ito et al. 1999).

According to Swartz's review paper (2005), it has been considered that physiological role in alkali-, Na and K "-resistance may secondarily impact processes such as sporulation and nitrogen fixation in particular species.

The Mrp antiporter system may have a substrate or activity in addition to the primary process/es, although still no clear result has been found yet.

4.2.4 Respiration-dependent ATP Synthesis

Alkaliphiles maintain a cytoplasmic pH lower than that of the outside pH. The proton motive force is about -50 mV at pH 10.5, which is the optimum pH value for many alkaliphiles, and this value is not sufficient to thrive in an alkaline milieu. Therefore, alkaliphiles must develop strategies for energy conservation. One possibility is to change the coupling ion, such as Na+-coupled ATPase. Krulwich and her colleagues reported, however, that respiratory chains that pump H + ion outward and H +-coupled ATP synthases are almost the same as these of neutrophilic microorganisms. Another possibility is that protons produced by electron transfer systems are not released into free solution but directly coupled to ATP synthase. Recently, Wang et al. (2004) showed that ATP synthesis at pH 10.5 in B. pseudofirmus OF4 depended upon alkaliphile-specific feature in the proton pathway through a- and c-subunits ATP synthase. They introduced site-directed changes in the a- and c subunits of the ATP synthase corresponding to the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATPsynthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH values. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Ap that are characteristic of alkaliphiles. These results strongly indicate that there are still many problems to be solved carefully. Further discussion on alkaliphily is conducted in a later section.

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