Stefanie Keis

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

ABSTRACT 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.

A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential

Despite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton‐coupled F1Fo‐ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c‐subunits using a novel mass spectrometry method, termed ‘laser‐induced liquid bead ion desorption’ (LILBID). This technique allows the mass determination of non‐covalently assembled, detergent‐solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.

A Clonal Lineage of VanA-Type Enterococcus faecalis Predominates in Vancomycin-Resistant Enterococci Isolated in New Zealand

ABSTRACT Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (≥256 μg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.

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.

ABSTRACT 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.

Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon 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 epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(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)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(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 epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon 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)(epsilon(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)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

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 (Deltap) 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 DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) 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 Deltap. 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 DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.

A Specific Adaptation in the a Subunit of Thermoalkaliphilic F1FO-ATP Synthase Enables ATP Synthesis at High pH but Not at Neutral pH Values

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F1Fo-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F1Fo) was developed in Escherichia coli DK8 (Δatp). Amino acid substitutions were made in the a subunit of TA2F1Fo at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus Pi-loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F1Fo and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0–9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys180 is a specific adaptation within the a subunit of TA2F1Fo to facilitate proton capture at high pH. At pH values near the pKa of Lys180, the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H+ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5′-GACA(N)7TGTC-3′, on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-d-maltoside, and subsequently purified it via Ni2+-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.

Nonfermentative Thermoalkaliphilic Growth Is Restricted to Alkaline Environments

ABSTRACT 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 F1Fo-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 F1Fo-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.

Draft Genome Sequence of the Thermoalkaliphilic Caldalkalibacillus thermarum Strain TA2.A1

The genes and molecular machines that allow for a thermoalkaliphilic lifestyle have not been defined. To address this goal, we report on the improved high-quality draft genome sequence of Caldalkalibacillus thermarum strain TA2.A1, an obligately aerobic bacterium that grows optimally at pH 9.5 and 65 to 70°C on a wide variety of carbon and energy sources.