Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

Succinate:quinone oxidoreductase (complex II) containing a single heme b in facultative alkaliphilic Bacillus sp. strain YN-2000.

Succinate:quinone oxidoreductase (EC 1.3.5.1) was first purified from the facultative alkaliphilic Bacillus sp. strain YN-2000 in the presence of Triton X-100. The isolated enzyme showed high succinate-ubiquinone oxidoreductase activity at pH 8.5. The Km for ubiquinone 1 and the Vmax of the enzyme were determined to be about 5 microM and 48 micromol of ubiquinone 1 per min per mg, respectively. The catalytic activity of the enzyme was 50% inhibited by 9 microM 2-thenoyltrifluoroacetone or 0.8 microM 2-n-heptyl-4-hydroxyquinoline- N-oxide. The enzyme consisted of three kinds of subunits with molecular masses of 66, 26, and 15 kDa, respectively, and contained 1.28 mol of covalently bound flavin adenine dinucleotide, 0.9 mol of heme b, 1.35 mol of menaquinone, 8.3 mol of nonheme iron, and 7.5 mol of inorganic sulfide per mol of enzyme. The enzyme showed symmetrical alpha absorption peaks at 556.5 and 554 nm in the reduced state at room temperature and 77 K, respectively. The potentiometric analysis of the enzyme yielded an Em,7 of heme b of about -64 mV (n = 1). Furthermore, the content of the enzyme was increased up to fivefold when the bacterium was grown at pH 10 compared with pH 7. These results indicate that the succinate:quinone oxidoreductase with a single heme b is involved in the respiratory chain of the alkaliphile at a very alkaline pH.     

The effect of salt on the lipid composition of Ectothiorhodospira

Major components of polar lipids of halophilic phototrophic Ectothiorhodospira species were PG, CL, PC and PE. PA was only present in minor amounts. According to ¹⁴C-incorporation, polar lipids approximated to 75%–93% of the total lipid carbon. With increasing salinity, a strong increase in the portion of PG and a decrease in that of PE (especially in Ectothiorhodospira mobilis BN 9903) and CL (especially in E. halophila strains) were observed. Moreover, there was a significant increase in the excess negative charges of phospholipids upon increasing medium salinity. This increase was most dramatic in the slightly halophilic E. mobilis BN 9903, but quantitatively less important in both strains of E. halophila which had, however, a higher percentage of negative charges of their lipids. During salt-shift experiments, E. halophila BN 9630 responded to suddenly increased salinity by promoting the biosynthesis of PG and decreasing that of PC, CL and PE. Upon dilution stress, responses were reversed and resulted in a strong increase in PE biosynthesis. The effects of lipid charges and bilayer forming forces in stabilizing the membranes of Ectothiorhodospira species during salt stress are discussed.Abbreviations PC phosphatidylcholine - PG, PG-1, PG-2 phosphatidylglycerol - CL, CL-1, Cl-2 cardiolipin - PE phosphatidylethanol-amine - PA phosphatidic acid - NL nonpolar lipids - ori origin - TLC thin layer chromatography     

Purification and characterization of catalase from a facultative alkalophilic Bacillus

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).     

Cardiolipin Is Dispensable for Oxidative Phosphorylation and Non-fermentative Growth of Alkaliphilic Bacillus pseudofirmus OF4

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from ³²P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28–33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.     

Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function

Cardiolipin (CL) is a unique phospholipid which is present throughout the eukaryotic kingdom and is localized in mitochondrial membranes. Saccharomyces cerevisiae cells containing a disruption of CRD1, the structural gene encoding CL synthase, have no CL in mitochondrial membranes. To elucidate the physiological role of CL, we compared mitochondrial functions in the crd1Delta mutant and isogenic wild type. The crd1Delta mutant loses viability at elevated temperature, and prolonged culture at 37 degrees C leads to loss of the mitochondrial genome. Mutant membranes have increased phosphatidylglycerol (PG) when grown in a nonfermentable carbon source but have almost no detectable PG in medium containing glucose. In glucose-grown cells, maximum respiratory rate, ATPase and cytochrome oxidase activities, and protein import are deficient in the mutant. The ADP/ATP carrier is defective even during growth in a nonfermentable carbon source. The mitochondrial membrane potential is decreased in mutant cells. The decrease is more pronounced in glucose-grown cells, which lack PG, but is also apparent in membranes containing PG (i.e. in nonfermentable carbon sources). We propose that CL is required for maintaining the mitochondrial membrane potential and that reduced membrane potential in the absence of CL leads to defects in protein import and other mitochondrial functions.     

Molar growth yields and bioenergetic parameters of extremely alkaliphilic Bacillus species in batch cultures, and growth in a chemostat at pH 10.5.

Alkaliphilic Bacillus species that grow at pH 10.5 must cope with a low protonmotive force (-50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mM buffered medium, while the actual medium pH remains above 10.2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10.4 and 10.6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7.5 or 10.5. Its molar growth yield (23 mg dry wt mmol-1) on malate (Ymal) was the same at the two pH values and was comparable to Ymal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10.5 also showed Ymal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7.5 (45.2 kJ mol-1) or pH 10.5 (46 kJ mol-1) was in a conventional range for bacteria.     

Study of interaction between cation fluxes in gramicidin A-modified bilayer phospholipid membranes. Determination of the number of ion-binding sites and their position in the gramicidin channel

Simultaneous studies were carried out of isotope and electric parameters of spheric bilayer membranes modified with gramicidin A and its analog O-pyromellithylgramicidin (PG) having three negative charges on the N-end of the molecule. The relationship between the electric coefficients of permeability and the isotope ones PG/P* = n was determined by two independent methods. It has been found that for the membranes modified with gramicidin A in RbCl concentrations from 2.2 x 10(-3) to 10(-1) M the value n is constant and approximates 2 and with RbCl concentration 1 M, n = 1.6. For the membranes modified with PG in 0.1 M solutions of PbCl n = 2. The results obtained in terms of the model of unilinear ion diffusion in a narrow pore indicate that in a gramicidin channel there are two sites of cation binding which are located near the channel mouth.     

Transfer of Tn925 and plasmids between Bacillus subtilis and alkaliphilic Bacillus firmus OF4 during Tn925-mediated conjugation.

Conjugative transposon Tn925 was transferred to alkaliphilic Bacillus firmus OF4 during mating experiments, as monitored by the acquisition of tetracycline resistance at pH 7.5 and confirmed by Southern analysis of chromosomal DNA from transconjugants. Tetracycline resistance could not be demonstrated at pH 10.5, but transconjugants retained resistance upon growth at pH 7.5 after having grown for several generations at pH 10. When the Bacillus subtilis donor strain contained plasmids, either pUB110 or pTV1, in addition to Tn925, transfer of the plasmid to the alkaliphile occurred during conjugation, either together with or independently of the transfer of the transposon. The plasmids were stable in B. firmus OF4, expressing their resistance markers for kanamycin or chloramphenicol at pH 7.5 after growth of the transformants at high pH. Transconjugant B. firmus OF4, which carried Tn925, could serve as the donor in mating experiments with B. subtilis lacking the transposon. These studies establish a basis for initiation of genetic studies in this alkaliphilic Bacillus species, including the introduction of cloned genes and the use of transposon-mediated insertional mutagenesis.     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4.

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.     

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production.     

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

Cloning and sequencing of the gene encoding a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na(+)-ATPase, Na(+)/K(+)-ATPase, H(+)-ATPases and sarcoplasmic reticulum Ca(2+)-ATPase, it exhibited the highest homology with Ca(2+)-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.     

Localization of Anionic Phospholipids in Escherichia coli Cells

Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.     

Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution

Sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) are lipids with negative charges, distributed among membranes of chloroplasts of plants and their postulated progenitors, cyanobacteria, and also widely among membranes of anoxygenic photosynthetic bacteria. Thus, these acidic lipids are of great interest in terms of their roles in the function and evolution of the photosynthetic membranes. The physiological significance of these lipids in photosynthesis has been examined through characterization of mutants defective in their abilities to synthesize SQDG or PG, and through characterization of isolated thylakoid membranes or photosynthetic particles, the acidic lipid contents of which were manipulated in vitro, for example, on treatment with phospholipase to degrade PG. Responsibility of SQDG or PG has been clarified so far in terms of the structural and/or functional integrity of photosystems I and/or II in cyanobacterial, green algal, and higher plant species. Also implied were distinct levels of the responsibility in the different photosynthetic organisms. Extreme cases involved the indispensability of SQDG for photosynthesis and growth in two prokaryotic, photosynthetic organisms and the contribution of PG to construction of the photosystem-I trimer exclusively in cyanobacteria. Here, roles of these acidic lipids are discussed with a focus on their specificity and the evolution of photosynthetic membranes.Norihiro Sato is the recipient of the Botanical Society Award for Young Scientist, 2003.     

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH?>?9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B.?pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.     

Nonfermentative Thermoalkaliphilic Growth Is Restricted to Alkaline Environments

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F₁F_o-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F₁F_o-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.Alkaliphilic microorganisms have been isolated from a diverse range of environments and have traditionally been classified into two distinct groups based on their pH profile for growth (8). Bacteria that grow across a broad pH range from 7.0 to 11.0 have been classified as facultative alkaliphiles (e.g., Bacillus pseudofirmus OF4) (28), and those that are able to grow only above pH 9.0 have been classified as obligate alkaliphiles (e.g., Bacillus alcalophilus) (4). The reasons why obligate alkaliphiles fail to grow below pH 9.0 remain speculative.While the classification of alkaliphilic bacteria based on pH profiles for growth has gained universal acceptance, it does not consider the nature of the carbon source that is used to grow the cells, and for aerobic alkaliphiles, this may have important consequences. For example, growth on succinate in aerobic bacteria is strictly coupled to oxidative phosphorylation and ATP is produced in the cell via the membrane-bound F₁F_o-ATP synthase. Growth on fermentable carbon sources, such as glucose, allows the cells to bypass this machinery, as ATP can be produced via substrate-level phosphorylation and incomplete oxidation of glucose to acetate can occur.A thermoalkaliphilic bacterium, Bacillus sp. strain TA2.A1, capable of optimal aerobic growth at a temperature of 65°C at pH 9.5 was isolated from an alkaline thermal bore at Mt. Te Aroha, New Zealand (19). The 16S rRNA gene sequence of strain TA2.A1, compared with those available in the EMBL database, shows 99.5% similarity to Caldalkalibacillus thermarum strain HA6^T, an aerobic, heterotrophic, thermophilic bacterium isolated from an alkaline hot spring in China (30). On the basis of the similarity of its phenotypic and genotypic characteristics to those of strain HA6^T, we assign strain TA2.A1 to the genus and species Caldalkalibacillus thermarum. C. thermarum strain TA2.A1 grows on sucrose, common C₄-dicarboxylates, glutamate, pyruvate, and trehalose; however, glucose and fructose fail to support growth (19). We originally described strain TA2.A1 as a facultative alkaliphile based on its pH profile for growth on glutamate or sucrose (18-20); however, both are substrates whose metabolism is not strictly coupled to oxidative phosphorylation.In this communication, we determine the pH profile for growth of C. thermarum strain TA2.A1 on nonfermentable (i.e., succinate and malate) and fermentable carbon sources (i.e., sucrose) using pH-controlled batch culture and demonstrate that strain TA2.A1 was unable to grow below pH 9.0 in pH-controlled batch culture on succinate but grew from pH 7.5 to 10 on sucrose. The physiological and biochemical bases for this phenomenon were investigated.     

Alkaliphilic heliobacterium Heliorestis baculata sp. nov. and emended description of the genus Heliorestis

A rod-shaped heliobacterium motile by peritrichous flagella, designated strain OS-H1, was isolated from a sample of shoreline soil of the soda lake Ostozhe (pH 9.2, total salt content 0.22%) located in the steppe of south-east Siberia. In the first few transfers, the isolate produced heat-resistant endospores. Like other heliobacteria, strain OS-H1 contained bacteriochlorophyll g and lacked intracytoplasmic membranes. The new isolate was a strict anaerobe and photoheterotroph. In the light and in the presence of organic compounds, strain OS-H1 oxidized sulfide to elemental sulfur and polysulfides, but was not capable of photoautotrophic growth. The isolate was an obligate alkaliphile able to grow at pH 8-10.2. The best growth was observed at pH 8.5-9.5, a temperature of 30 degrees C and at 5-10 g sodium carbonate l(-1). Biotin was required as a growth factor. The G+C content of strain OS-H1 was 45.0 mol%. Comparison of the 16S rRNA gene sequence to that of phototrophic bacteria showed strain OS-H1 to group within gram-positive bacteria of the family Heliobacteriaceae with the closest relationship to Heliorestis daurensis (95.6% similarity). Based on physiological, genetic and chemotaxonomic characteristics, the new heliobacterium is described as a new species of the genus Heliorestis, Heliorestis baculata.     

Surface charges on chloroplast membranes as studied by particle electrophoresis.

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts. 2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge. 3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface. 4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues. 5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting chlorophyll a/chlorophyll b pigment . protein complex. 6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts. 7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory. 8. Calculations of the zeta-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500--2000 A2. 9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.