BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance

Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.     

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance

The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro-phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the Importance of various regions in transport.     

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.     

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.     

Resistance to Bacitracin as Modulated by an Escherichia coli Homologue of the Bacitracin ABC Transporter BcrC Subunit from Bacillus licheniformis

A small open reading frame from the Escherichia coli chromosome, bcrC(EC), encodes a homologue to the BcrC subunit of the bacitracin permease from Bacillus licheniformis. We show that disruption of the chromosomal bcrC(EC) gene causes bacitracin sensitivity and, conversely, that BcrC(EC) confers bacitracin resistance when expressed from a multicopy plasmid.     

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.     

Accumulation of heptaprenyl diphosphate sensitizes Bacillus subtilis to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter

Heptaprenyl diphosphate (C₃₅‐PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C₃₅‐PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C₅₅‐PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C₃₅‐PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C₃₅‐PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C₃₅‐PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepT or by supplementation with 1,4‐dihydroxy‐2‐naphthoate, a co‐substrate with C₃₅‐PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C₃₅‐PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C₃₅‐PP inhibits both BcrC (a C₅₅‐PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C₅₅‐PP, rather than the antibiotic across the membrane.     

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.     

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.     

Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.     

Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two extracytoplasmic function sigma factors

Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C(55)-isoprenyl phosphate by de novo synthesis. We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant. A bcrC mutant strain had an eightfold-higher sensitivity to bacitracin. Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) sigma factors, sigma(X) or sigma(M). Bacitracin induced expression of bcrC, and this induction was dependent on sigma(M) but not on sigma(X). Under inducing conditions, expression was primarily dependent on sigma(M). As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive. A sigX sigM double mutant was similar to a bcrC mutant in sensitivity. These results support the suggestion that one function of B. subtilis ECF sigma factors is to coordinate antibiotic stress responses.     

Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.     

代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先,构建了l-半胱氨酸合成酶基因cysK强化表达菌株,杆菌肽效价相比于对照菌株提高了9.47%。接着,为提高l-半胱氨酸合成前体供给,对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化,杆菌肽产量分别提高了7.23%和8.52%。随后,结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸,强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种,在整合表达了基因cysK基础上,利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子,得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP)),杆菌肽效价达到910.02 U/mL,相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后,通过3 L发酵罐小试实验,进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明,强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略,为杆菌肽工业生产提供了一株具有良好应用前景的菌株。     

Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.

An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification. One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance. The bacA gene mapped to approximately 67 min on the E. coli chromosome by proximity to a previously mapped locus. The deduced amino acid sequence of the bacA-encoded protein suggests an extremely hydrophobic protein of 151 amino acids, approximately 65% of which were nonpolar amino acids. E. coli cells containing plasmid pXV62 have increased isoprenol kinase activity. The physical characteristics of the deduced protein and enhanced lipid kinase activity suggest that the bacA gene may confer resistance to bacitracin by phosphorylation of undecaprenol.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.