Identification of RR binding sites

Upstream regions of operons encoding Pep7E ABC transporters were retrieved from the MicrobesOnline database (Dehal et al., 2010). Generally, 250 nucleotides (nt) upstream of the start codons were analyzed,

unless the distance to the nearest upstream gene was greater than 250 bp. In this case, the whole intergenic region was analyzed. If the gene encoding the ATPase subunit was not the first in the predicted operon, both 250 bp upstream of the ATPase gene and the region upstream of the preceding gene were analyzed. Retrieved sequences were subjected to conserved motif searches using MEME (Bailey and Elkan, 1994) (http://meme.sdsc.edu/) with the following parameters: distribution, any number of repetitions; width, minimum of 5, maximum of 20. Analysis with MEME was followed by in-depth manual analysis. Sequence motifs were illustrated based on a position weight matrix using the WebLogo tool (Crooks et al., 2004)

(http://weblogo.berkeley.edu).

4.5. Acknowledgments

We thank Ralf Zimmer for valuable input regarding calculations of correlation coefficients for multiple sequence alignments.

Work done in our laboratory was supported by grants-in-aid from the Fonds der Chemischen Industrie (to S.G.) and a grant from the Deutsche Forschungsgemeinschaft (to T.M.; grant MA2837/1-3).

CHAPTER 5

Peptide antibiotic sensing and detoxification modules

of Bacillus subtilis

This chapter has been adapted from:

A. Staroń, D. E. Finkeisen, T. Mascher

Chapter 5

Peptide antibiotic sensing and detoxification modules of B. subtilis

Peptide antibiotics are produced by a wide range of microorganisms. Most of them target the cell envelope, often by inhibiting cell wall synthesis. One of the resistance mechanisms against antimicrobial peptides is a detoxification module consisting of a two-component system and an ABC transporter. Upon the detection of such a compound, the two- component system induces the expression of the ABC transporter, which in turn removes the antibiotic from its site of action, mediating the resistance of the cell. Three such peptide antibiotic-sensing and detoxification modules are present in B. subtilis. Here we show that each of these modules responds to a number of peptides and confers resistance against them. BceRS-BceAB (BceRS-AB) responds to bacitracin, plectasin, mersacidin, and actagardine. YxdJK-LM is induced by a cationic antimicrobial peptide, LL-37. The PsdRS- AB (formerly YvcPQ-RS) system responds primarily to lipid II-binding lantibiotics such as nisin and gallidermin. We characterized the psdRS-AB operon and defined the regulatory sequences within the PpsdA promoter. Mutation analysis demonstrated that PpsdA

expression is fully PsdR dependent. The features of both the PbceA and PpsdA promoters

make them promising candidates as novel whole-cell biosensors that can easily be adjusted for high-throughput screening.

5.1. Introduction

Peptide antibiotics are produced by a wide range of organismsand can be synthesized both ribosomally and nonribosomally (Jenssen et al., 2006). Nonribosomally synthesized antimicrobial compounds areproduced mainly by bacteria and are often posttranslationally modified (Hancock and Chapple, 1999). They can form linear polypeptides, such as gramicidin (Killian, 1992), or cyclic molecules, such as bacitracin and polymyxins (Landman et al., 2008; Ming, 2003). Glycopeptides (e.g., vancomycin and ramoplanin) consistof a peptide backbone, which is further modified by glycosylationand methylation (Donadio and Sosio, 2008). Ribosomally synthesized peptides, includinglantibiotics and defensins, are more widespread and are producedby mammals, amphibians, insects, plants, and bacteria (Hancock and Chapple, 1999).They are often derived from small precursor peptides and areusually small (10 to 50 amino acids), with an overall positivecharge and a significant number of hydrophobic residues (Hancock and Sahl, 2006).

Most peptide antibiotics target crucial steps in cell wall biosynthesis. The bacterial cell wall is a vitally important structure that gives the cell its shape, separates it from its environment,and acts as a molecular sieve (Jordan et al., 2008). This makes it an important target for many antimicrobial compounds, which very often actby sequestering lipid II and by blocking transglycosylation and transpeptidation steps (Schneider and Sahl, 2010). Vancomycin, lantibiotics, ramoplanin,and many defensins bind different moieties of lipid II (Jordan et al., 2008; Schmitt et al., 2010; Schneider et al., 2010). Vancomycin binds to the C-terminal Lys-D-Ala-D-Ala of the pentapeptide chain of the cell wall precursor

(Breukink and de Kruijff, 2006). Nisinand nisin-like lantibiotics bind the pyrophosphate of lipidII, whereas the binding site of mersacidin and related lantibiotics includes both the MurNAc-GlcNAc sugar moiety and the pyrophosphate (Cudic et al., 2002). Ramoplanin requires the presence of MurNAc-Ala-Glu pyrophosphate in order to bind to lipid II. Bacitracin inhibits a different step of cell wall biosynthesis by binding undecaprenyl pyrophosphate and inhibiting its dephosphorylation, thereby blocking its recycling and, ultimately, cell wall biosynthesis (Rietkötter et al., 2008).

Because the production of peptide antibiotics is widespread,the presence of an appropriate stress response system is necessary both for the producer strains as well as for those bacteria that are exposed to these compounds in their natural habitat. One type of detoxification system against peptide antibioticsfound mainly in Gram-positive bacteria is a module consistingof an ABC transporter, which is genetically and functionallylinked to a 2CS (Jordan et al., 2008; Joseph et al., 2002; Mascher, 2006). Upon sensingthe signal (i.e., the presence of the antibiotic), the HK phosphorylates its cognate RR, whichin turn induces the expression of the ABC transporter genes. The transporter facilitates the removal of the antibiotic compoundfrom its active site (Jordan et al., 2008).

While few of these systems have been experimentally characterizedto date, all respond and mediate resistance to cell wall peptideantibiotics. In S. aureus, the GraRS-VraFG system was previously found to respond to vancomycin, polymyxin B (Meehl et al., 2007), gallidermin (Herbert et al., 2007), and defensins (Kraus et al., 2008). Homologous proteins mediate resistance to nisin in L. lactis (Kramer et al., 2006) and tobacitracin in S. mutans (Ouyang et al., 2010; Tsuda et al., 2002).

The genome of B. subtilis contains three such peptide-sensing and detoxification (PSD) modules consisting of a 2CS and an ABC transporter: BceRS-AB, YxdJK-LM, and YvcPQ-RS (Fig. 5.1 Aand B). BceRS-AB (PSD1) was initially identified as a bacitracin- specificdetoxification module (Mascher et al., 2003; Ohki et al., 2003a). Recently, it was

also shownto respond to the defensin plectasin (Schneider et al., 2010). The YxdJK-LM system (PSD2) responds to the human antimicrobial peptide LL-37 (Pietiäinen et al., 2005).The third system, YvcPQ-RS (PSD3), was initially described asa part of bacitracin stress response network (Mascher et al., 2003).

Figure 5.1. Organization of the psdRS-AB and bceRS-AB

loci and induction by peptide antibiotics. (A) Graphic representation of the psdRSAB and bceRSAB loci. Genes

belonging to the psd and bce loci are shown in blue (two-

component system) and green (ABC transporter); the genes flanking both operons are white. Promoters are marked with bent arrows, and putative terminators are represented by vertical bars and a circle. (B) Regulatory principle and genetic setup of the Psd and Bce biosensor strains. The RR (PsdR or BceR), activated by the sensor kinase (PsdS or BceS), binds to its target promoter and induces the expression of the ABC transporter encoding the psdAB or bceAB operon (detoxification) and lacZ

(production of β-galactosidase). (C) Example of a

qualitative β-galactosidase assay with nisin, actagardine,

mersacidin, and gramicidin (disk diffusion assay). The reporter strain carrying a chromosomal PpsdA-lacZ fusion

was used in soft-agar overlays on LB plates containing X-Gal. Bactericidal activity is visualized as the presence of a growth inhibition zone around the filter disk, and PpsdA-dependent induction is visualized as a blue ring

around the inhibition zone. (D) Qualitative β-

galactosidase assay with the subtilin producer strain B. subtilis ATCC 6633. PpsdA-lacZ reporter strain TMB299

was streaked out onto LB-X-Gal plates directly next to the B. subtilis ATCC 6633 cultures. The appearance of a

blue zone in the reporter strain next to the subtilin- producing strain shows the induction of PpsdA.

In this study, we aimed to identify novel inducers for all threePSD modules. Using disk diffusion assays and promoter-lacZ fusions,we screened a wide variety of cell envelope-active compounds, including many peptide antibiotics. In addition, we performed a comprehensive meta-analysis of all previously published stressresponse microarray data sets in order to identify additional inducers of bceAB, yvcRS, and yxdLM expression.

We present evidence that the BceRS-AB system is not only a bacitracin-specificresistance determinant but rather a PSD module that respondsto a broader spectrum of compounds, including the lantibioticsmersacidin and actagardine as well as the defensin plectasin.This module also mediates a certain level of resistance to thesecompounds.

For PSD3, it was recently shown that the weak induction of the yvcR promoter by bacitracin is the result of a cross-activationof the RR YvcP by the HK of theparalogous

BceRS-AB system (Rietkötter et al., 2008). In this study, we identifiedlipid II-binding peptides, mainly lantibiotics but also onelipopeptide, enduracidin, as inducers of yvcRS expression. We further demonstrate that the ABC transporter YvcRS confers resistance against its inducers. Based on the primary inducers and theresistance profile, we renamed the yvcPQRS locus to psdRSAB(for peptide antibiotic sensing and detoxification).

Our data demonstrate that the PbceA- and PpsdA-based reporterstrains are more sensitive and

more specific biosensors forlipid II-binding peptide antibiotics than any of the established cell wall antibiotic biosensors currently available, such as the PypuA- and PliaI-derived

reporter strains (Mascher et al., 2004; Urban et al., 2007). We provideevidence indicating that both biosensors could easily be modifiedto accommodate high-throughput screens for novel antimicrobial compounds using pure compounds, culture supernatant, or even, directly, the producing strains.

5.2.Results and discussion