Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Thermophilic enzymes can operate at higher temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule F\"orster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.Comment: 14 pages, 7 figure

Taxonomy and phylogeny of industrial solvent-producing clostridia

xii, 314 leaves :ill. (some col.) ; 30 cm. Includes bibliographical references. "June 1996". Includes previously published material by the author. University of Otago department: Microbiology.Solvent-producing clostridial strains were used extensively during the first part of this century for the industrial production of acetone and butanol and in the last two decades have again become the focus of investigation due to their potential applications in biotechnology. A starch-fermenting Clostridium species, isolated and patented during World War I and later named Clostridium acetobutylicum, was the first successful industrial strain to be used for the large-scale production of solvents. From the mid-1930's onwards, when molasses became abundant, the acetone-butanol (AB) fermentation process in most countries was switched from utilizing starch-based substrates to this Jess expensive sugar-based substrate. This in turn led to the isolation and patenting of numerous solvent-producing clostridia capable of fermenting molasses, each of which was given a novel species name. However, the majority of these saccharolytic solvent-producing clostridial strains were never scientifically recognized as legitimate species and once the industrial AB fermentation process declined these names fell into disuse. At present the majority of the industrial solvent-producing clostridia held in culture collections around the world tend to be classified as C. acetobutylicum or C. beijerinckii. In recent years however, there has been the growing awareness that there is a considerable degree of heterogeneity amongst strains currently classified as C. acetobutylicum. This has highlighted the need for a re-examination and re-assessment of the taxonomic and phylogenetic relationships of this industrially important group of bacteria. In this study, 72 solvent-producing clostridial strains, the majority of which are currently classified as C. acetobutylicum, were examined by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. DNA fingerprinting was achieved by digesting genomic bacterial DNA with infrequently cutting restriction endonucleases and resolving the resulting fragments by pulsed-field gel electrophoresis (PFGE). Based on the results obtained from these two approaches the 72 strains could be divided into 17 distinct groups. There was a high degree of correlation between the biotypes and DNA fingerprints within each group indicating that the strains were similar both phenotypically and genotypically. The DNA fingerprints obtained for strains belonging to the same group exhibited high levels of similarity but differed markedly between the 17 groups. To establish the relationships of the different groups, the partial 16S rRNA gene sequence, corresponding to positions 830-1383 (Escherichia coli numbering), of a prototype strain from each group was determined. This was achieved by polymerase chain reaction (PCR) amplification and direct sequencing of the resultant PCR product using primers designed in this study. DNA sequence analysis of this segment of the 16S rRNA gene provided a relatively quick and reliable method of comparing the prototype strains. The results obtained of a comparative analysis of the partial 16S rRNA gene sequences, indicated that the 17 biotype and DNA fingerprint groups could be assembled into four taxonomic groups (taxonomic groups I-IV). In order to establish more definitive phylogenetic positions for the four taxonomic groups of solvent-producing clostridia, the complete 16S rRNA gene sequences were determined for the strains ATCC 824(T), NCP 262, Nl-4, and NCIMB 8052 which represented the taxonomic groups I, II, III, and IV, respectively. The phylogenetic analysis of the complete 16S rRNA gene sequences revealed that amylolytic strains belonging to taxonomic group I were only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90%- 90.5%). The NCIMB 8052 strain (taxonomic group IV), formerly catalogued as being equivalent to the C. acetobutylicum type strain ATCC 824(T) (taxonomic group I), was found to belong to a different species based on 16S rRNA gene sequence analysis. The NCIMB 8052 strain exhibited 100% level of 16S rRNA gene sequence similarity with the type strain of C. beijerinckii. The strains belonging to taxonomic groups II, III, and IV were much more closely related (levels of sequence similarity, 98.2%- 98.9%). Based on the 16S rRNA gene sequences alone it was not possible to determine whether these three taxonomic groups of saccharolytic solvent-producing clostridia constituted three separate species or were three subgroups belonging to a single species since they exhibited sequence similarities higher than 97%. However, during the course of this study the results obtained by Johnson and Chen (1995) from genomic DNA-DNA hybridization studies of solvent-producing clostridia, established that the three taxonomic groups II, III, and IV were all separate species of saccharolytic solvent-producing clostridia. Their analysis also supports the finding that taxonomic group I consisted of a collection of distantly related amylolytic strains originally designated C. acetobutylicum. The outcome of this study and that of other researchers has indicated that all of the saccharolytic strains presently classified as C. acetobutylicum will need to be reclassified. The names "C. saccharo-butyl-acetonicumliquefaciens," "C. saccharoperbuty/acetonicum," and C. beijerinckii are proposed for the three saccharolytic solvent-producing Clostridium species on the basis of prior usage

Biochemical and Molecular Characterization of a Na(+)-Translocating F(1)F(o)-ATPase from the Thermoalkaliphilic Bacterium Clostridium paradoxum

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56°C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F(1)F(o)-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q(28), E(61), and S(62)). On the basis of these properties, the F(1)F(o)-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of Na(+) that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the ɛ subunit that has been shown to be essential for coupled ATP synthesis

Inhibition of ATP Hydrolysis by Thermoalkaliphilic F(1)F(o)-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ɛ subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated ɛ subunit [i.e., TA2F(1)(ɛ(ΔC))] or substitution of basic residues in the second α-helix of ɛ with nonpolar alanines [i.e., TA2F(1)(ɛ(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(ɛ(WT)) complex with proteases revealed that the ɛ subunit was resistant to proteolytic digestion. In contrast, the ɛ subunit of TA2F(1)(ɛ(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ɛ was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ɛ subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(ɛ(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(ɛ(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the ɛ subunit also inhibits ATPase activity of TA2F(o)F(1)

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme

The Structural Basis for Unidirectional Rotation of Thermoalkaliphilic F1-ATPase

SummaryThe ATP synthase of the thermoalkaliphilic Bacillus sp. TA2.A1 operates exclusively in ATP synthesis direction. In the crystal structure of the nucleotide-free α3β3γɛ subcomplex (TA2F1) at 3.1 Å resolution, all three β subunits adopt the open βE conformation. The structure shows salt bridges between the helix-turn-helix motif of the C-terminal domain of the βE subunit (residues Asp372 and Asp375) and the N-terminal helix of the γ subunit (residues Arg9 and Arg10). These electrostatic forces pull the γ shaft out of the rotational center and impede rotation through steric interference with the βE subunit. Replacement of Arg9 and Arg10 with glutamines eliminates the salt bridges and results in an activation of ATP hydrolysis activity, suggesting that these salt bridges prevent the native enzyme from rotating in ATP hydrolysis direction. A similar bending of the γ shaft as in the TA2F1 structure was observed by single-particle analysis of the TA2F1Fo holoenzyme

Bioenergetic Properties of the Thermoalkaliphilic Bacillus sp. Strain TA2.A1

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Δp) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum ΔpH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Δψ) was maintained between −114 mV and −150 mV, and little significant change was observed over the pH range for growth. In contrast, the Δp declined from −164 mV at pH 7.5 to approximately −78 mV at pH 10.0. An inwardly directed sodium motive force (ΔpNa(+)) of −100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Δp. The phosphorylation potential of strain TA2.A1 was maintained between −300 mV and −418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the ΔpNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Δp. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium

Characterization of a Vancomycin-Resistant Enterococcus faecalis (VREF) Isolate from a Dog with Mastitis: Further Evidence of a Clonal Lineage of VREF in New Zealand

We report here on the characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolated from a dog with mastitis. The isolate was positive for the vanA, ermB, and tet(M) genes, with vanA and ermB carried on the same transferable plasmid. Comparison of this isolate with VREF from poultry and human sources in New Zealand demonstrated identical SmaI macrorestriction patterns and Tn1546-like elements. This is further evidence of a clonal lineage of VREF in New Zealand

Acquired Bacitracin Resistance in Enterococcus faecalis Is Mediated by an ABC Transporter and a Novel Regulatory Protein, BcrR

Bacitracin resistance (bacitracin MIC, ≥256 μg ml(−1)) has been reported in Enterococcus faecalis, and in the present study we report on the genetic basis for this resistance. Mutagenesis was carried out with transposon Tn917 to select for E. faecalis mutants with decreased resistance to bacitracin. Two bacitracin-sensitive mutants (MICs, 32 μg ml(−1)) were obtained and Tn917 insertions were mapped to genes designated bcrA and bcrB. The amino acid sequences of BcrA (ATP-binding domain) and BrcB (membrane-spanning domain) are predicted to constitute a homodimeric ATP-binding cassette (ABC) transporter, the function of which is essential for bacitracin resistance in E. faecalis. The bcrA and bcrB genes were organized in an operon with a third gene, bcrD, that had homology to undecaprenol kinases. Northern analysis demonstrated that bcrA, bcrB, and bcrD were transcribed as a polycistronic message that was induced by increasing concentrations of bacitracin but not by other cell wall-active antimicrobials (e.g., vancomycin). Upstream of the bcrABD operon was a putative regulatory gene, bcrR. The bcrR gene was expressed constitutively, and deletion of bcrR resulted in a bacitracin-sensitive phenotype. No bcrABD expression was observed in a bcrR mutant, suggesting that BcrR is an activator of genes essential for bacitracin resistance (i.e., bcrABD). The bacitracin resistance genes were found to be located on a plasmid that transferred at a high frequency to E. faecalis strain JH2-2. This report represents the first description of genes that are essential for acquired bacitracin resistance in E. faecalis