The role of the bacitracin ABC transporter in bacitracin resistance and collateral detergent sensitivity

The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr. Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein. Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC. A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed. It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.     

Bacitracin sensing in Bacillus subtilis

The extracellular presence of antibiotics is a common threat in microbial life. Their sensitive detection and subsequent induction of appropriate resistance mechanisms is therefore a prerequisite for survival. The bacitracin stress response network of Bacillus subtilis consists of four signal-transducing systems, the two-component systems (TCS) BceRS, YvcPQ and LiaRS, and the extracytoplasmic function (ECF) σ factor σ^M. Here, we investigated the mechanism of bacitracin perception and the response hierarchy within this network. The BceRS–BceAB TCS/ABC transporter module is the most sensitive and efficient bacitracin resistance determinant. The ABC transporter BceAB not only acts as a bacitracin detoxification pump, but is also crucial for bacitracin sensing, indicative of a novel mechanism of stimulus perception, conserved in Firmicutes bacteria. The Bce system seems to respond to bacitracin directly (drug sensing), whereas the LiaRS TCS and σ^M respond only at higher concentrations and indirectly to bacitracin action (damage sensing). The YvcPQ–YvcRS system is subject to cross-activation via the paralogous Bce system, and is therefore only indirectly induced by bacitracin. The bacitracin stress response network is optimized to respond to antibiotic gradients in a way that maximizes the gain and minimizes the costs of this stress response.     

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.     

Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes Bacteria

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.     

Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC(90) (>256 μg/ml) was identical for both turkey and chicken isolates; whereas MIC(50) was higher in turkey isolates (6 μg/ml) than in chicken isolates (3 μg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 μg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens.     

Bacitracin sensing and resistance in Staphylococcus aureus

Bacterial two-component systems (TCSs) have been demonstrated to be associated with not only the expression of virulence factors, but also the susceptibility to antibacterial agents. In Staphylococcus aureus, 16 types of TCSs have been identified. We previously found that the inactivation of one uncharacterized TCS (designated as BceRS, MW gene ID: MW2545-2544) resulted in an increase in susceptibility to bacitracin. In this study, we focused on this TCS and tried to identify the TCS-controlled factors affecting the susceptibility to bacitracin. We found that two ABC transporters were associated with the susceptibility to bacitracin. One transporter designated as BceAB (MW2543-2542) is downstream of this TCS, while another (formerly designated as VraDE: MW2620-2621) is separate from this TCS. Both transporters showed homology with several bacitracin-resistance factors in Gram-positive bacteria. Inactivation of each of these two transporters increased the susceptibility to bacitracin. Expressions of these transporters were significantly increased by the addition of bacitracin, while this induction was not observed in the TCS-inactivated mutant. These results indicate that this TCS senses bacitracin, and also positively regulates the expression of two ABC transporters.     

The BceRS two-component regulatory system induces expression of the bacitracin transporter,BceAB, in Bacillus subtilis

BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis. Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus. Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium. The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB. Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR. The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes. The bcrC gene product is additionally involved in bacitracin resistance. The expression of bcrC is dependent on the ECF sigma factors, sigmaM and sigmaX, but not on the BceRS two-component system. In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.     

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis

Abstract Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.     

Identification of various substrate-binding proteins of the hyperthermophylic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

Proteins from the extracellular medium of Aeropyrum pernix K1 were separated by two-dimensional electrophoresis and identified using mass spectrometry. Six different substrate-binding proteins (SBPs) from the ATP-binding cassette (ABC) transporter family were identified: (1) ABC transporter SBP (Q9YC61); (2) Branched-chain amino-acid ABC transporter, branched-chain amino-acid-binding protein (Q9YDJ6); (3) Oligopeptide ABC transporter, oligopeptide-binding protein (Q9YBL5); (4) Probable ABC transporter SBP (Q9Y9N4); (5) ABC transporter SBP (Q9YBG7); (6) ABC transporter SBP (Q9YFD7). Based on their orthology, division into the following classes was predicted: (1) multiple sugar-transport system SBPs; (2) peptide/nickel-transport system SBPs; and (3) branched-chain amino-acid-transport system SBPs. Further bioinformatic analyses showed that the identified SBPs differ in motif and in transmembrane-domain and signal-peptide organisation. Additionally, for all of these SBPs, sequence homology was found for archaeal proteins, and homologous proteins in bacteria were also found for the ABC transporter SBP Q9YBG7 and the ABC transporter SBP Q9YFD7. This is the first study, where different ABC SBPs from the extracellular medium of A. pernix have been identified using the combined methodology of two-dimensional electrophoresis and mass spectrometry.     

The two-component regulatory system BacRS is associated with bacitracin 'self-resistance' of Bacillus licheniformis ATCC 10716.

Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram-positive microorganisms, but not against the producer strain itself. Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described. In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44-kb bacitracin biosynthetic operon bacABC. Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified. They code for proteins with high homology to regulator and sensor proteins of two-component systems. A disruption mutant of the bacRS genes was constructed. While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild-type strain. Western blot analysis of the expression of BcrA, the ATP-binding cassette of the transporter, showed in the wild-type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive. A comparison of bacitracin stressed and nonstressed wild-type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells. From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes.     

假单胞菌M18株pltZ基因转录阻抑藤黄绿菌素ABC转运系统

假单胞菌(Pseudomonassp .)M18株的藤黄绿菌素(Pyoluteorin ,Plt )生物合成基因簇下游存在一个Plt生物合成负调控基因pltZ和一个负责Plt分泌及自身抗性的ABC(ATP_bindingcassette)转运系统基因簇。利用启动子探针载体pME6 0 15和pME6 5 2 2分别构建ABC转运基因pltH与lacZ的翻译和转录融合表达质粒pHZLF和pHZCF ,分别引入野生型假单胞菌M18株和pltZ突变菌株M18Z。半乳糖苷酶活性的测定结果表明:在pltZ突变株M18Z中,pltH’-‘lacZ翻译融合表达水平约比野生型提高3 7～8 4倍,pltH’‘lacZ转录融合表达水平显著提高了2 8～7 4倍,表明pltZ能在转录水平上阻抑PltABC转运系统的表达,pltZ很可能通过阻抑PltABC转运系统的表达,间接地负调控Plt的生物合成     

Anatomy of the bacitracin resistance network in Bacillus subtilis

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well‐characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle‐inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage‐shock proteins LiaIH. Our systems‐level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.     

A new highly conserved antibiotic sensing/resistance pathway in firmicutes involves an ABC transporter interplaying with a signal transduction system

Signal transduction systems and ABC transporters often contribute jointly to adaptive bacterial responses to environmental changes. In Bacillus subtilis, three such pairs are involved in responses to antibiotics: BceRSAB, YvcPQRS and YxdJKLM. They are characterized by a histidine kinase belonging to the intramembrane sensing kinase family and by a translocator possessing an unusually large extracytoplasmic loop. It was established here using a phylogenomic approach that systems of this kind are specific but widespread in Firmicutes, where they originated. The present phylogenetic analyses brought to light a highly dynamic evolutionary history involving numerous horizontal gene transfers, duplications and lost events, leading to a great variety of Bce-like repertories in members of this bacterial phylum. Based on these phylogenetic analyses, it was proposed to subdivide the Bce-like modules into six well-defined subfamilies. Functional studies were performed on members of subfamily IV comprising BceRSAB from B. subtilis, the expression of which was found to require the signal transduction system as well as the ABC transporter itself. The present results suggest, for the members of this subfamily, the occurrence of interactions between one component of each partner, the kinase and the corresponding translocator. At functional and/or structural levels, bacitracin dependent expression of bceAB and bacitracin resistance processes require the presence of the BceB translocator loop. Some other members of subfamily IV were also found to participate in bacitracin resistance processes. Taken together our study suggests that this regulatory mechanism might constitute an important common antibiotic resistance mechanism in Firmicutes. [Supplemental material is available online at http://www.genome.org.].