Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Oligonucleotides used in this study.
Figure 1.
Stained flagellar of B. alcalophilus and alignment with flagella motor sequences from other bacteria.
Alignments of the region containing the single transmembrane segment of E. coli MotB (EC_MotB), B. subtilis MotB (BS_MotB) and MotS (BS_MotS), B. licheniformis MotB (BL_MotB) and MotS (BL_MotS), Geobacillus kaustophilus MotB (GK_MotB), Oceanobacillus iheyensis MotB (OI_MotB) and MotS (OI_MotS), B. clausii MotB (BCl_MotB), B. alcalophilus MotS (BA_MotS), B. pseudofirmus MotS (BP_MotB), B. halodurans MotS (BH_MotB), B. megaterium MotB (BM_MotS), V. alginolyticus MotB (VA_MotB) and PomB (VA_PomB), V. parahaemolyticus MotB (VP_MotB) and PomB (VP_PomB), V. mimicus MotB (VM_MotB), V. splendidus PomB (VS_PomB), and V. fisheri PomB (VF_PomB). The position of D32 in EC_MotB is known to be critical for rotation and is highlighted in green. The MotAB of B. clausii can use Na+ instead of H+ to promote flagellar rotation at high pH values. The V37L mutation was critical for sodium selectivity and a combination of the V37L mutation and either the A40S or the G42S mutation was required for production of the BCl-MotB (the ninth line) form that exhibits sodium-coupling at low pH [14]. The position of V43 in EC_MotB (the first line) is conserved among all of the MotB-H+-type proteins and is highlighted in light blue. The position of L32 in BP_MotS (the eleventh line from the top) is conserved among all of the MotS-Na+-type proteins with the exception of BA_MotS and is highlighted in yellow. The same position in B. alcalophilus MotS encodes methionine instead of the conserved leucine residue, and it is highlighted with violet.
Figure 2.
Growth of neutralophilic B. subtilis two alkaliphilic Bacillus species.
(A), (B) and (C) show the growth data for B. subtilis, B. alcalophilus and B. pseudofirmus, respectively. Growth in MYE medium (pH 7.5, red filled circle and pH 10.5, red open circle) and KMYE medium (pH 7.5, light-blue filled circle and pH 10.5, light-blue open circle) at 30°C was monitored at A600. The results are the averages of three independent experiments, with error bars representing the standard deviations.
Figure 3.
Motility of B. pseudofirmus and B. alcalophilus in liquid medium.
B. pseudofirmus OF4 and B. alcalophilus cells in the logarithmic growth phase that were grown at 30°C in MYE medium (pH 10.5) and KMYE medium (pH 10.5), respectively, were harvested and resuspended in 30 mM Tris-HCl (pH 9.0) that contained 5 mM glucose and the indicated sodium (A), potassium (B) or rubidium (C) concentrations as described in the Materials and Methods section. The red line and red open circles show the data for the B. pseudofirmus OF4 strain, and the blue line and blue filled circles show the data for the B. alcalophilus strain. The swimming speed was determined as described in the Materials and Methods section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.
Figure 4.
The swimming speed of two alkaliphiles dependent upon pH and concentrations of NaCl and KCl.
The relationship between the swimming speed and several different pH values at 200 mM Na+ (A) or K+ (B) is illustrated. The relationship between swimming speed in 30 mM Tris-HCl containing 5 mM glucose (pH 9.0) and the various indicated concentrations of KCl and NaCl is shown in (C). The red line and red open circles show the data for the B. pseudofirmus OF4 strain, and the blue line and blue filled circles show the data for the B. alcalophilus strain. The swimming speed was determined as described in the Materials and Methods section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.
Figure 5.
Effect of the Na+ channel inhibitor EIPA on motility.
The red line and red filled circles show the data for the B. pseudofirmus strain, and the blue line and blue filled circles show the data for the B. alcalophilus strain in Tris-HCl buffer that contained 5 mM glucose and 200 mM NaCl (pH 9.0) (A) or 200 mM KCl (pH 9.0) (B). The swimming speed was determined as described in the Materials and Methods. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.
Figure 6.
The effect of KCl and NaCl on swimming speed of B. subtilis and E.coli strains.
The effect of KCl and NaCl on swimming speed of B. subtilis strains (A) and (B), The velocity was measured at several different pH values in phosphate buffer that contained 200 mM Na+, 150 mM Na+ plus 50 mM K+, 100 mM Na+ plus 100 mM K+, 50 mM Na+ plus 150 mM K+, or 200 mM K+. The blue line and blue filled circles, the green line and green filled circles, and the red line and red filled circles show the data at pH 7.0, 7.5, and 8.0, respectively. The effect of KCl and NaCl on swimming speed of E. coli strain (C) The velocity was measured at pH 7.0 in phosphate buffer that contained 200 mM Na+, 150 mM Na+ plus 50 mM K+, 100 mM Na+ plus 100 mM K+, 50 mM Na+ plus 150 mM K+, or 200 mM K+. The blue line and blue filled circles and the red line and red filled circles show the data for EC-BAPS and EC-BAPS-MotS_M33L, respectively. The swimming speed was determined as described in the Materials and Methods section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.
Figure 7.
Effect of KCl on the growth and intracellular ion content of various E. coli TK2420 transformants.
The growth of E. coli strain DH5αMCR transformed with control plasmid pBAD24 (filled blue circles) and E. coli strain TK2420 transformed with pBAD24 (open blue circles), pBAPS (filled red circles) and pBAPS-MotS_M33L (open red circles). Cells were shaken in the TK2420 minimal medium adding 10 mM (A), 25 mM (B) or 50 mM (C) KCl at 37°C under aerobic conditions. Cell growth was monitored at 600 nm. Intracellular [K+] and [Na+] levels in E. coli DH5αMCR transformed with control plasmid pBAD24 (filled light blue bar) and E. coli strain TK2420 transformed with pBAD24 (open light blue filled bar), pBAPS (filled red bar) and pBAPS-MotS_M33L (open red bar). Cells were shaken in the TK2420 minimal medium adding 25 mM (D), or 50 mM (E) KCl at 37°C under aerobic conditions. The results are the averages of three independent duplicate experiments, with error bars representing the standard deviations.
Figure 8.
The Na+ and K+ cycles of alkaliphiles.
A diagrammatic illustration of the pH homeostasis capacity of the Na+- and Na+ plus K+-dependent alkaliphiles and elements of their membrane-associated Na+, K+ and H+ translocation pathways.