gebhard abc transporters of antimicrobial peptides in the firmicutes

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MicroReview ABC transporters of antimicrobial peptides in Firmicutes bacteria – phylogeny, function and regulation Susanne Gebhard* Ludwig-Maximilians-Universität München, Department Biology I, Microbiology, Grosshaderner Str. 2-4, 82152 Planegg-Martinsried, Germany. Summary Antimicrobial peptides (AMPs) are a group of antibiot- ics that mainly target the cell wall of Gram-positive bacteria. Resistance is achieved by a variety of mecha- nisms including target alterations, changes in the cell’s surface charge, expression of immunity pep- tides or by dedicated ABC transporters. The latter often provide the greatest level of protection. Apart from resistance, ABC transporters are also required for the export of peptides during biosynthesis. In this review the different AMP transporters identified to date in Firmicutes bacteria were classified into five distinct groups based on their domain architecture, two groups with a role in biosynthesis, and three involved in resistance. Comparison of the available information for each group regarding function, transport mecha- nism and gene regulation revealed distinguishing characteristics as well as common traits. For example, a strong correlation between transporter group and mode of gene regulation was observed, with three different types of two-component systems as well as XRE family transcriptional regulators commonly asso- ciated with individual transporter groups. Further- more, the presented summary of the state-of-the-art on AMP transport in Firmicutes bacteria, discussed in the context of transporter phylogeny, provides insights into the mechanisms of substrate translocation and how this may result in resistance against compounds that bind extracellular targets. Introduction As bacterial infections are becoming increasingly difficult to treat due to rising numbers of multidrug-resistant strains, efforts are being directed towards the discovery of new drugs, ideally targeting different bacterial structures to circumvent the development of cross-resistance. One group of antibiotics that has received considerable atten- tion in recent years are the antimicrobial peptides (AMPs). They include structurally very diverse compounds, such as the heavily modified lantibiotics (e.g. nisin or mersacidin), unmodified bacteriocins (e.g. pediocin), non-ribosomally synthesized cyclic AMPs (e.g. bacitracin), glycopeptides (e.g. vancomycin) or lipodepsipeptides (e.g. ramoplanin) (reviewed for example in Ennahar et al., 2000; Guder et al., 2000; Cotter et al., 2005; Breukink and de Kruijff, 2006). Their target spectra can also differ considerably, but most of the compounds mentioned are active against Gram-positive bacteria with a low G+C content (Firmi- cutes). All of these AMPs share a mode of action that inhibits the lipid II cycle of cell wall biosynthesis, albeit at different steps. Bacteriocins of class I (lantibiotics) and class II (non-lantibiotic bacteriocins) bind to lipid II, prevent- ing incorporation of cell wall precursors into the growing peptidoglycan layer. Some bacteriocins are able to form pores in the cytoplasmic membrane, often using lipid II as a docking molecule (Guder et al., 2000; Cotter et al., 2005; Breukink and de Kruijff, 2006). Ramoplanin and enduraci- din were shown to interfere with the transglycosylation reaction of cell wall biosynthesis, again by binding to lipid II (Fang et al., 2006), while vancomycin binds the terminal D-Ala–D-Ala of the pentapeptide chain, thus inhibiting transpeptidation (Perkins, 1969). Finally, bacitracin binds to undecaprenyl-pyrophosphate (UPP) that remains after incorporation of the cell wall precursors and thereby pre- vents recycling of the lipid carrier molecule undecaprenyl- phosphate (UP) (Storm and Strominger, 1973). To defend themselves against AMPs, Firmicutes bacte- ria have developed a number of different mechanisms. For example vancomycin resistance is achieved by changing the terminal D-Ala residue of the pentapeptide chain to Accepted 16 October, 2012. *For correspondence. E-mail [email protected]; Tel. (+49) (0) 89 2180 74659; Fax (+49) (0) 89 2180 74626. Molecular Microbiology (2012) 86(6), 1295–1317 doi:10.1111/mmi.12078 First published online 19 November 2012 © 2012 Blackwell Publishing Ltd

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Page 1: Gebhard ABC Transporters of Antimicrobial Peptides in the Firmicutes

MicroReview

ABC transporters of antimicrobial peptides in Firmicutesbacteria – phylogeny, function and regulation

Susanne Gebhard*Ludwig-Maximilians-Universität München, DepartmentBiology I, Microbiology, Grosshaderner Str. 2-4, 82152Planegg-Martinsried, Germany.

SummaryAntimicrobial peptides (AMPs) are a group of antibiot-ics that mainly target the cell wall of Gram-positivebacteria. Resistance is achieved by a variety of mecha-nisms including target alterations, changes in thecell’s surface charge, expression of immunity pep-tides or by dedicated ABC transporters. The latteroften provide the greatest level of protection. Apartfrom resistance, ABC transporters are also requiredfor the export of peptides during biosynthesis. In thisreview the different AMP transporters identified to datein Firmicutes bacteria were classified into five distinctgroups based on their domain architecture, twogroups with a role in biosynthesis, and three involvedin resistance. Comparison of the available informationfor each group regarding function, transport mecha-nism and gene regulation revealed distinguishingcharacteristics as well as common traits. For example,a strong correlation between transporter group andmode of gene regulation was observed, with threedifferent types of two-component systems as well asXRE family transcriptional regulators commonly asso-ciated with individual transporter groups. Further-more, the presented summary of the state-of-the-art onAMP transport in Firmicutes bacteria, discussed in thecontext of transporter phylogeny, provides insightsinto the mechanisms of substrate translocation andhow this may result in resistance against compoundsthat bind extracellular targets.

IntroductionAs bacterial infections are becoming increasingly difficultto treat due to rising numbers of multidrug-resistant strains,efforts are being directed towards the discovery of newdrugs, ideally targeting different bacterial structures tocircumvent the development of cross-resistance. Onegroup of antibiotics that has received considerable atten-tion in recent years are the antimicrobial peptides (AMPs).They include structurally very diverse compounds, such asthe heavily modified lantibiotics (e.g. nisin or mersacidin),unmodified bacteriocins (e.g. pediocin), non-ribosomallysynthesized cyclic AMPs (e.g. bacitracin), glycopeptides(e.g. vancomycin) or lipodepsipeptides (e.g. ramoplanin)(reviewed for example in Ennahar et al., 2000; Guderet al., 2000; Cotter et al., 2005; Breukink and de Kruijff,2006).

Their target spectra can also differ considerably, butmost of the compounds mentioned are active againstGram-positive bacteria with a low G+C content (Firmi-cutes). All of these AMPs share a mode of action thatinhibits the lipid II cycle of cell wall biosynthesis, albeit atdifferent steps. Bacteriocins of class I (lantibiotics) andclass II (non-lantibiotic bacteriocins) bind to lipid II, prevent-ing incorporation of cell wall precursors into the growingpeptidoglycan layer. Some bacteriocins are able to formpores in the cytoplasmic membrane, often using lipid II asa docking molecule (Guder et al., 2000; Cotter et al., 2005;Breukink and de Kruijff, 2006). Ramoplanin and enduraci-din were shown to interfere with the transglycosylationreaction of cell wall biosynthesis, again by binding to lipid II(Fang et al., 2006), while vancomycin binds the terminalD-Ala–D-Ala of the pentapeptide chain, thus inhibitingtranspeptidation (Perkins, 1969). Finally, bacitracin bindsto undecaprenyl-pyrophosphate (UPP) that remains afterincorporation of the cell wall precursors and thereby pre-vents recycling of the lipid carrier molecule undecaprenyl-phosphate (UP) (Storm and Strominger, 1973).

To defend themselves against AMPs, Firmicutes bacte-ria have developed a number of different mechanisms. Forexample vancomycin resistance is achieved by changingthe terminal D-Ala residue of the pentapeptide chain to

Accepted 16 October, 2012. *For correspondence. [email protected]; Tel. (+49) (0) 89 2180 74659; Fax(+49) (0) 89 2180 74626.

Molecular Microbiology (2012) 86(6), 1295–1317 ! doi:10.1111/mmi.12078First published online 19 November 2012

© 2012 Blackwell Publishing Ltd

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D-Lac, which drastically reduces binding by vancomycin(Arthur and Courvalin, 1993). Many AMPs are positivelycharged, and a general resistance mechanism can there-fore be a reduction in the net negative charge of thebacterial cell wall. One way this is achieved is by incorpo-ration of D-alanine into teichoic acids, thus adding a posi-tive charge. D-alanylation of teichoic acids is catalysed bythe products of the dltABCDE operon (Neuhaus and Bad-diley, 2003; Cao and Helmann, 2004; McBride and Sonen-shein, 2011). Another mechanism is lysinylation of themembrane lipid phosphatidylglycerol, catalysed by MprF,which also adds a positive charge to the cell envelope(Ernst and Peschel, 2011). Producer strains of lantibioticsoften possess specific immunity peptides, collectivelyreferred to as LanI, which function in self-protection(Draper et al., 2008). However, the most efficient resist-ance mechanisms often involve ATP-binding cassette(ABC) transporters. Several different types of such trans-porters have been described as self-resistance mecha-nisms in AMP producing strains as well as for protectionagainst foreign AMPs. In addition to providing resistance,ABC transporters are also required for peptide exportduring biosynthesis of AMPs.

A number of excellent reviews have summarized thebiosynthesis, structure and mode of action of AMPs(Ennahar et al., 2000; Guder et al., 2000; Cotter et al.,2005; Breukink and de Kruijff, 2006; Draper et al., 2008;Kjos et al., 2011; Alkhatib et al., 2012). However, no dedi-cated article has been published on the various ABCtransporters involved in AMP export, self-immunity andresistance. The aims of this review were therefore to collectthe wealth of information published to date on such trans-porters from Firmicutes bacteria, to derive a classificationthat can be easily applied to newly identified transporters,and to compare the identified groups regarding their physi-ological role, transport mechanism and gene regulation.

Classification of AMP transporters based ondomain architectureAs mentioned above, several different types of ABC trans-porters for AMPs have been identified in Firmicutes bac-teria. Annotation of newly identified transporter genes ishereby usually based on sequence similarity to previouslydescribed systems. One aim of this review was to pool thecurrently available functional information on these trans-porters to facilitate more informed predictions. While thedatabases contain many ABC transporters that either areencoded in AMP biosynthetic loci or have been implicatedin resistance from physiological experiments, there is asyet no convincing classification. A search of the availableliterature showed that AMP transporters from Firmicutescould be divided into five groups by a simple comparisonof their predicted domain architecture using the SMART

database (Letunic et al., 2012). These proposed groups,each named after one characterized member, can bedescribed as follows.

SunT-type transporters

This group contains large transporters of approximately700 amino acids with the ATPase domain fused to theC-terminus of the permease domain (Fig. 1A, dark blueframe, and Fig. 4A). Additionally, they contain anN-terminal peptidase C39 domain, which is responsible forpre-peptide processing. Their permease domains consistof five or six predicted transmembrane helices, dependingon the employed algorithm. Available functional datasuggest an intracellular localization of the peptidasedomain, thus six transmembrane helices appear morelikely as discussed below (Håvarstein et al., 1995; Frankeet al., 1999). The SMART database (Letunic et al., 2012)contains over 800 proteins with this composition (January2012), mostly from Firmicutes and Proteobacteria, but alsofrom Actinobacteria and Cyanobacteria. After removal ofduplicates, 181 distinct SunT-type transporters could beidentified in Firmicutes bacteria (Table S1).

NisT-type transporters

These proteins are similar to SunT-type transportersexcept that they do not contain a peptidase domain andare thus smaller with approximately 550–600 amino acids(Fig. 1A, purple frame, and Fig. 4B). It is not feasible toperform database searches for these transporters basedon either domain architecture or sequence similarityalone, because they resemble prototypical fused ABCtransporters and therefore produce large numbers of hitswith unrelated function. A search of the transport classifi-cation database (TCDB) (Saier et al., 2009), supple-mented with a manual search of known AMP biosyntheticloci for transporters with the correct domain architectureresulted in a list of nine NisT-type proteins (Table S1).

LanFEG-type transporters

This group contains transporters with two separate per-meases of 200–250 amino acids and six transmembranehelices each and one ATPase (Fig. 1A, green frame, andFig. 4C). A homology-based analysis of all available bac-terial genomes in the MicrobesOnline database (Dehalet al., 2010) using the ‘tree’ search function and theNukFEG transporter of Staphylococcus warneri (Okudaet al., 2008) as query, supplemented with known LanFEGtransporters, produced a list of 29 systems (Table S1).

BceAB-type transporters

These transporters are comprised of two proteins, oneATPase and one permease of approximately 650 amino

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acids and 10 transmembrane helices with a large (c. 200amino acid) extracellular domain located between helicesVII and VIII (Fig. 1A, yellow frame, and Fig. 4D). Helices IIto IV form an FtsX domain, which can be used as query indomain-based searches of the SMART database (Letunicet al., 2012). An earlier study had shown the existence ofover 260 such transporters in genomes of Firmicutes bac-teria (Dintner et al., 2011) (see Table S1 for 24 examplesequences).

BcrAB-type transporters

Systems from this group consist of one permease ofapproximately 230 amino acids with six predicted trans-membrane helices and one ATPase (Fig. 1A, lightblue/red frame, and Fig. 4E). As with the NisT-typetransporters, a domain-based database search was notfeasible. However, a search of all available bacterialgenomes based on sequence similarity to the per-meases of two well-characterized members, BcrB ofBacillus licheniformis (Podlesek et al., 1995) and YydIof Bacillus subtilis (Butcher et al., 2007), using BLASTP

and the MicrobesOnline database (Altschul et al., 1990;Dehal et al., 2010) resulted in a list of 18 such trans-porters (Table S1).

Classification and phylogeny of AMP transporters

Phylogenetic trees calculated for the ATPase compo-nents of all transporters (see supplemental text S1 fordetails on the phylogenetic analyses) showed that theproteins largely clustered according to the domain-basedclassification described above (Fig. 1A), even though thelatter was mainly based on differences in the permeases.An analysis of the permease regions resulted in a similarphylogenetic tree (Fig. 1B). It should be noted that theBceAB-type transporters were not included in the secondanalysis, because their 10-transmembrane-helix per-meases could not be aligned well with the six-helixpermeases of the remaining transporter groups (see sup-plemental text S1). Comparison of the phylogenetic treesto the domain-based classification described aboveshowed that the BceAB and LanFEG groups of trans-porters formed distinct phylogenetic clusters. The SunTand NisT transporters formed one intermixed group,suggesting that presence or absence of the peptidasedomain does not reflect the evolutionary relationshipbetween both groups. Further, the BcrAB-type transport-ers fell into two branches according to sequence similar-ity either to BcrB of B. licheniformis (red lines) or to YydIof B. subtilis (light blue lines). It therefore appears rea-sonable to split this group into two subfamilies, based onsequence similarity. A striking observation was that theBcrAB-like transporters were found to be closely related

and ancestral to the LanFEG-type transporters. Thelatter appear to have originated from a duplication ofthe permease gene, followed by divergence of the twonew permeases (dark green lines for LanF, light greenlines for LanG). The observed mirror-image of the LanFand LanG clusters suggested a co-evolution of the twosubunits within each transporter (Pazos and Valencia,2008).

Overall, the domain-based classification of AMP trans-porters described here presents a fast and convenienttool for the analysis of newly identified transporters andaccurately reflects the phylogeny of these protein families.Therefore the nomenclature suggested above will beused throughout this review.

Genomic context of AMP transporter genesAntimicrobial peptide (AMP) transporters have oftenreceived only cursory attention during the characterizationof novel biosynthesis loci, mainly regarding their role inproducer self-immunity or in export of the synthesizedpeptides. Therefore limited experimental data are avail-able on their precise function, transport mechanism oreven regulation. Often, valuable functional informationabout a gene can be derived from the conservation of itsgenomic context. A detailed analysis of the neighbouringgenes of a subset of transporters from each group showedthat, while rarely two loci had an identical arrangement,a correlation between transporter type and associatedgenes nevertheless existed (Fig. 2 and Table 1). Func-tional categories included for example structural genes forAMPs and their modification, transcriptional regulators orimmunity proteins. It is important to keep in mind that likelymany functionally important genes have been missedbecause of low sequence conservation, particularly ofAMP structural or immunity genes. This may be reflectedin the large number of small hypothetical genes, often withone or more predicted transmembrane helices, found inthe neighbourhood of the transporter genes (Table 1).With the exception of BcrAB- and BceAB-type transport-ers, the transporter loci also often contained transposasesor other mobility-associated elements, indicating a highdegree of horizontal gene transfer (Table 1). It should benoted that genomic context was analysed only for a fewrandomly selected example transporters from each groupand any numbers given refer to this limited subset. In thefollowing sections each type of transporter is reviewedregarding their physiological role, transport mechanismand regulation, connected where possible to a discussionof genomic context conservation. For reasons of concise-ness the main focus was placed on systems that havebeen at least partially characterized. A summary of alltransporters discussed, including their substrate peptides,is given in Table 2.

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Fig. 2. Genomic context of AMP transporters. For each transporter group three examples (shown boxed) were chosen to reflect a range ofobserved genomic context arrangements. The category of each gene is colour coded as detailed at the bottom of the figure. Transmembranehelices of hypothetical proteins or XRE regulators are shown by black vertical lines; those of histidine kinases (HK) are indicated by verticalmedium blue lines. Sizes of genes and intergenic regions not to scale. RR, response regulator, Pep.-QS, peptide quorum sensing; Peripl.,periplasmic sensing; IM, intramembrane-sensing; Tran. acc., transport accessory protein; BL, biotinyl-lipoyl domain.

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SunT-type transportersPhysiological role

The transporters of the SunT group are involved in exportof AMPs or peptide pheromones with concomitantcleavage of the leader peptides (Håvarstein et al., 1995).However, analysis of the genomic context of 40 exampletransporters showed that only about half were actuallyassociated with AMP biosynthetic genes (Table 1).Whether this means that the remaining transporters trulyexist in the absence of such genes or is simply due to poorgene annotation is difficult to answer. AMP transport by aSunT-type transporter has for example been demonstratedfor MutT (mutacin II), LctT (lacticin 481), LcnC (lactococcinA), PedD (pediocin PA-1), AvcT (avicin A) and LcaC (leu-cocin A) (Marugg et al., 1992; Stoddard et al., 1992; vanBelkum and Stiles, 1995; Birri et al., 2010). Experimentalevidence for export is usually presented as absence of thetarget AMP in culture supernatants of transporter mutants.Additionally, it was shown that cells harbouring transport-deficient mutants of NukT accumulated the nukacinprecursor peptide intracellulary, demonstrating a lack ofpeptide export (Nishie et al., 2011). Interestingly, the SunTgroup does not only contain exporters for AMPs, but alsofor small peptides used for quorum sensing by Gram-positive bacteria. The best-characterized example is theComA transporter, which is involved in competence regu-lation in Streptococcus pneumoniae and is required forexport of the competence stimulating peptide (CSP) ComC

(Hui et al., 1995). A second example is SilE of Streptococ-cus pyogenes, which is responsible for excretion of theautoinducer peptide SilCR required for invasive infections(Hidalgo-Grass et al., 2002).

Transport mechanism

Many SunT-type transporters were found to be associatedwith transport accessory proteins (Table 1). Experimentalevidence that these proteins are required for export ofsubstrate peptides by the ABC transporter is available forseveral examples, including LcnD (for LcnC) (Stoddardet al., 1992; Franke et al., 1999) and ComB (for ComA)(Hui et al., 1995). The transport accessory proteinspossess one predicted transmembrane helix with theN-terminus located intracellulary and the bulk of theprotein extracellularly, as was confirmed experimentallyfor LcnD (Franke et al., 1996). The precise mechanism oftransport involving such accessory proteins is unknown.Some accessory proteins discovered during the genomiccontext analysis, including LcnD, were considerablylarger than others (about 450 compared with about 170amino acids) and contained an additional biotinyl-lipoyldomain of unknown function (Fig. 2). Conversely, morethan half of the SunT-type transporters analysed here lackan obvious accessory protein (Table 1), including MutT,MrsT, NukT, ScnT and SunT. Export of the bacteriocinssakacin T and sakacin X by StxT, which also lacks anaccessory protein, was experimentally shown (Vaughan

Table 1. Occurrence of genes by functional categories in genomic context of AMP transporters.

Gene categorya SunT (40) NisT (9) LanFEG (30) BceAB (23) BcrAB (13) YydIJ (5)

Peptide quorum sensor TCS 18% – – – – –Periplasmic sensing TCS 13%b 44%b 73% – – –Intramembrane-sensing TCS 3%c – 10%c 78% 62% –XRE regulator 15% 33% 3% – 31% (23%)d –Other regulators 8% 11% 7% 13% – 20%SunT/NisT-like – – 28% – – –LanFEG-like 28% 56% 3%e – – –BceAB-like 5% 11% 10% 13%e – –Transport accessory 43% – – – – –Peptidase/protease 5% 67% 3% 4% – 40%Bacteriocin/toxin 58% 100% 31% – – 20%Modification 55% 89% 28% – – 20%Immunity 20% 78% 10% – – –UppP-like – – – 4% 77% –Transposase/mobility 33% 22% 24% 4% 8% 20%1–4 TM HP 25% 33% 41% 22% 15% 40%

a. Genes found in each locus sorted by functional category; distributions are given as per cent of loci containing at least one gene in each category,a dash indicates no such gene found; the total number of loci analysed for each transporter type is given in parentheses in the title row. See bodytext for detail on classification of TCSs; 1–4 TM HP, hypothetical protein with one to four predicted transmembrane helices; for more details seebody text.b. All these loci also contain a NisFEG-like transporter.c. All these loci also contain a BceAB-like transporter.d. Number in parentheses shows loci with an XRE regulator containing four transmembrane helices at the N-terminus.e. Two or more transporters of the same type found in one locus.

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Table 2. Summary of transporters discussed in this review.

Name Substrate Classa Organism References

SunT groupAvcT Avicin A Class IIa bacteriocin Enterococcus avium 208 Birri et al. (2010)BreH Brevicin 925A Class II bacteriocin Lactobacillus brevis 925A Wada et al. (2009)ComA CSP Quorum sensing Streptococcus pneumoniae Hui et al. (1995)LagD Lactococcin G Class IIb bacteriocin Lactococcus lactis LMG 2081 Håvarstein et al. (1995)LcaC Leucocin A Class IIa bacteriocin Leuconostoc gelidum UAL187 van Belkum and Stiles (1995)LcnC Lactococcin A Class IId bacteriocin Lactococcus lactis WM4 Stoddard et al. (1992); Franke et al. (1999)LctT Lacticin 481 Lantibiotic Lactococcus lactis IL1403 Uguen et al. (2005)MesD Mesentericin Y105 Class II bacteriocin Leuconostoc mesenteroides Y105 Fremaux et al. (1995)MrsT Mersacidin Lantibiotic Bacillus sp. HIL Y-85 Altena et al. (2000)MutT Mutacin II Lantibiotic Streptococcus mutans T8 Chen et al. (1999)NukT Nukacin Lantibiotic Staphylococcus warneri ISK-1 Aso et al. (2004)PedD Pediocin PA-1 Class IIa bacteriocin Pediococcus acidilactici PA-1 Marugg et al. (1992)PlnG Plantaricin 1.25b Class II bacteriocin Lactobacillus plantarum TMW1.25 Ehrmann et al. (2000)ScnT Streptococcin A-FF22 Lantibiotic Streptococcus pyogenes FF22 McLaughlin et al. (1999)SilE SilCR Quorum sensing Streptococcus pyogenes Hidalgo-Grass et al. (2002)SmbG SmbAB Dipeptide lantibiotic Streptococcus mutans GS5 Yonezawa and Kuramitsu (2005)StxT Sakacin T & X Class IIb & IIa bacteriocin

respectivelyLactobacillus sakei 5 Vaughan et al. (2003)

SunT Sublancin 168 Lantibiotic Bacillus subtilis W168 Paik et al. (1998)

NisT groupAurT Aureocin A70 Multipeptide bacteriocin Staphylococcus aureus A70 Netz et al. (2001)EpiT Epidermin Lantibiotic Staphylococcus epidermidis Tü3298 Peschel et al. (1997)EriT Ericin A & S Lantibiotic Bacillus subtilis A1/3 Stein et al. (2002a)GdmT Gallidermin Lantibiotic Staphylococcus gallinarum Tü3928 Peschel et al. (1997)LasT Lactocin S Lantibiotic Lactobacillus sakei L45 Skaugen et al. (2002)MutT Mutacin I Lantibiotic Streptococcus mutans CH43 and other

strainsQi et al. (2000)

NisT Nisin A Lantibiotic Lactococcus lactis N8 Qiao and Saris (1996); Kuipers (2004)NsuT Nisin U Lantibiotic Streptococcus uberis 42 Wirawan et al. (2006)PepT Pep5 Lantibiotic Staphylococcus epidermidis 5 Meyer et al. (1995)SpaT Subtilin Lantibiotic Bacillus subtilis ATCC6633 Klein et al. (1992)

LanFEG groupCprABC Nisin, Gallidermin Lantibiotics Clostridium difficile McBride and Sonenshein (2010)EpiFEG Epidermin, Gallidermin Lantibiotics Staphylococcus epidermidis Tü3298 Otto et al. (1998)LtnFE Lacticin 3147 Dipeptide lantibiotic Lactococcus lactis DPC3147 McAuliffe et al. (2001); Draper et al. (2009)McdFEG Macedocin Lantibiotic Streptococcus macedonicus ACA-DC 198 Papadelli et al. (2007)MrsFGE Mersacidin Lantibiotic Bacillus sp. HIL Y-85 Guder et al. (2002)NisFEG Nisin Lantibiotic Lactococcus lactis Ra et al. (1996); Stein et al. (2003)NukFEG Nukacin Lantibiotic Staphylococcus warneri ISK-1 Okuda et al. (2008); Okuda et al. (2010)SboFEG Salivaricin B Lantibiotic Streptococcus salivarius K12 Hyink et al. (2007)ScnFEG Streptococcin A-FF22 Lantibiotic Streptococcus pyogenes FF22 McLaughlin et al. (1999)SpaFEG Subtilin Lantibiotic Bacillus subtilis ATCC6633 Klein and Entian (1994); Stein et al. (2005)

BceAB groupAnrAB Nisin, gallidermin, bacitracin,

penicillin, (others)Lantibiotics, cyclic AMP,

b-lactam antibioticsListeria monocytogenes Collins et al. (2010)

BceAB Bacitracin, actagardine,mersacidin

Cyclic AMP, Lantibiotics Bacillus subtilis Ohki et al. (2003b); Rietkötter et al. (2008);Staron et al. (2011)

BraDE Bacitracin, nisin Cylic AMP, lantibiotic Staphylococcus aureus Blake et al. (2011); Hiron et al. (2011); Kolaret al. (2011); Yoshida et al. (2011)

MbrAB Bacitracin Cyclic AMP Streptococcus mutans Tsuda et al. (2002); Ouyang et al. (2010)PsdAB Nisin, subtilin, gallidermin,

enduracidinLantibiotics, glycodepsipeptide Bacillus subtilis Staron et al. (2011)

VraDE Bacitracin, nisin Cyclic AMP, Lantibiotic Staphylococcus aureus Pietiäinen et al. (2009); Hiron et al. (2011);Yoshida et al. (2011)

VraFG Nisin, colistin, bacitracin,vancomyin, indolicidin,LL-37, hBD3

Lantibiotic, cylic AMPs,glycopeptide, cathelicidins,defensin

Staphylococcus aureus Meehl et al. (2007); Falord et al. (2012)

YxdLM LL-37 Cathelicidin Bacillus subtilis Joseph (2004)

BcrAB/YydIJ groupBcrAB Bacitracin Cyclic AMP Bacillus licheniformis ATCC10716 Podlesek et al. (1995)BcrAB Bacitracin Cyclic AMP Enterococcus faecalis AR01/DGVS Manson et al. (2004)YydIJ YydF Peptide of unknown function Bacillus subtilis Butcher et al. (2007)

a. Where applicable, the classification according to Cotter et al. (2005) was applied; for reasons of simplicity, lantibiotics (class I bacteriocins) were only separated into single(‘Lantibiotic’) and dipeptide lantibiotics.

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et al., 2003), demonstrating that not all SunT-type trans-porters require such a protein.

A distinguishing feature of SunT-type transporters istheir possession of an N-terminal peptidase domain(Figs 1A and 4A), which is responsible for cleavage of theleader peptide. The N-terminal 150 amino acids of thelactococcin G transporter LagD, containing the predictedpeptidase domain, were shown to be sufficient for in vitrocleavage of the precursor peptide (Håvarstein et al., 1995).Similarly, the N-terminal domain of LcnC was shown to berequired for cleavage of pre-lactococcin A (Franke et al.,1999). An important question in this is whether the pepti-dase domain is located extra- or intracellularly. Predictionsof the transmembrane topology of SunT-type transportersare unclear: the in silico analyses carried out to derive theclassification presented above showed mainly five or sixtransmembrane helices, depending on the algorithm used.However, topology assays of LcnC using fusions to PhoAand LacZ suggested the existence of only four helices(Franke et al., 1999). Experimental data for LcnC andNukT show cytoplasmic localization of the peptidasedomain (Franke et al., 1999; Nishie et al., 2011), thus aneven number of transmembrane helices, whether four orsix, has to be assumed. Such an arrangement would alsobe consistent with the proposal that substrate recognitionby the transporter occurs via peptide binding to the proteo-lytic domain (Håvarstein et al., 1995). Interestingly, ATPhydrolysis by theATPase domain and leader peptide cleav-age by the peptidase domain are closely coupled coopera-tive activities during substrate processing and transport,that is mutants of NukT in either domain were incapableboth of transport and of leader cleavage (Nishie et al.,2011).

An early observation suggested a correlation betweenthe sequence of leader peptide and type of exporter:substrates with a leader sequence of the double-glycinetype are exported by large transporters with a fusedpeptidase domain, while those with unrelated leadersequences are exported by smaller transporters (Håvar-stein et al., 1995; Paik et al., 1998). Applying this obser-vation to the classification presented above, SunT- andNisT-type transporters should recognize substrates withdiffering leader sequences. An alignment of the first 30amino acids of eight substrate peptides each for SunT-and NisT-type transporters showed an excellent agree-ment with this proposition (Fig. 3): all SunT-type sub-strates possessed the GG/GS motif for cleavage,preceded by a conserved EL/ES motif, while none of theNisT-type substrates contained these elements. Impor-tantly, this applied to all SunT-type substrates tested,including lantibiotics (e.g. sublancin, mutacin II), class IIAMPs (e.g. pediocin, lactococcin A) or quorum sensingpeptides (e.g. ComC, SilCR), as noted previously (Paiket al., 1998).

Regulation

The expression of SunT-type transporters appears mainlyto be regulated in context with the surrounding biosyn-thetic gene clusters. Analysis of regulatory genes in thegenomic neighbourhood of these transporters showedthat many were associated either with a two-componentsystem (TCS) consisting of a six-transmembrane-helixhistidine kinase belonging to the group of peptide quorumsensors (Mascher et al., 2006) and a LytR familyresponse regulator, or with an XRE-type transcriptionalregulator (Table 1, Fig. 4A). In a few cases a differenttype of TCS was found, which can be explained by thepresence of additional AMP transporters in the samelocus, as discussed below.

Peptide quorum sensor TCSs were named based on therole of some characterized members in quorum sensing byGram-positive microorganisms (Mascher et al., 2006). Forexample the histidine kinase ComD of S. pneumoniaeinduces competence in response to the CSP peptide, anda connection between quorum sensing and competenceregulation has been drawn (Morrison and Lee, 2000).Similarly, SilB of S. pyogenes responds to the autoinducerpeptide SilCR to induce expression of the streptococcalinvasion locus (Hidalgo-Grass et al., 2002; Eran et al.,2007). Both CSP and SilCR are first exported by a SunT-type transporter, whose expression is also induced by theTCS in the presence of the signalling peptides (Morrisonand Lee, 2000; Eran et al., 2007). Export of the peptide isessential for gene regulation, because mutations in the

Fig. 3. Leader sequences of substrate peptides of SunT-type andNisT-type transporters. CLUSTALW alignment of the first 30 aminoacids of SunT-type substrate peptides (A) and NisT-type substratepeptides (B). Identical residues are boxed, similar residues (70%similarity) are shaded.

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corresponding SunT-type transporter abolish signalling,which can be restored by externally added peptide (Huiet al., 1995; Eran et al., 2007). Stimulus detection by thesequorum sensing kinases appears to involve non-specificinteractions of the inducing peptide with a hydrophobicpocket formed by the transmembrane helices, followed bysequence-specific interactions with one of the kinase’sextracellular loops (Mascher et al., 2006).

Regulation of AMP biosynthetic loci containing a SunT-type transporter and associated peptide quorum sensorkinase appears to follow the same principle. For example,expression of the avicin A production locus is induced bybinding of the pheromone peptide AvcP to the histidinekinase AvcT (Birri et al., 2010), and expression ofsakaxinX/T production is regulated via binding of thepeptide StxP to the histidine kinase StxK (Vaughan et al.,2003). Many biosynthetic loci containing a SunT-typetransporter and a peptide quorum sensor TCS lack adesignated inducing peptide. It is however possible thatregulation of these loci is autoinduced by the presence ofthe encoded AMP itself. Analyses of the promoters regu-lated by the peptide quorum sensor TCSs revealed thepresence of conserved binding sites consisting of 9 or10 bp direct repeats with a spacing of 11 or 12 bp (Belot-serkovsky et al., 2009; Birri et al., 2010).

The high similarity in regulation of quorum sensing,competence and AMP production, together with the con-servation of transporter type used for peptide export indi-cates a common evolutionary origin, or even developmentof one feature out of the other. This close connection isnicely exemplified by the Smb biosynthetic locus of Strep-tococcus mutans: it does not contain any regulatorygenes of its own, but is regulated by quorum sensing inresponse to the CSP peptide (Yonezawa and Kuramitsu,2005). A sequence with high similarity to the com-box wasidentified upstream of the smb operon, thus regulationmay occur via the alternative sigma factor for quorumsensing, ComX (Morrison and Lee, 2000; Yonezawa andKuramitsu, 2005).

Some SunT-type transporters were not found associ-ated with a TCS but instead with an XRE-type transcrip-tional regulator (Table 1, Fig. 4A). For example, expres-sion of the mutacin II biosynthetic operon mutAMTFEG isregulated by the XRE regulator MutR (Qi et al., 1999), anda mutR-disrupted strain was found to produce no mutacinII (Chen et al., 1999). It is thought that MutR responds tosome component of complex growth media to inducemutacin II production, but the precise signal has not beenidentified (Qi et al., 1999). Because these regulators do notpossess any transmembrane helices, they most likelydetect an intracellular stimulus, in contrast to the extracel-lular sensing by TCSs. The brevicin 925A biosyntheticlocus also contains an XRE regulator, BreG, but its role ingene regulation has not been investigated (Wada et al.,

2009). The presence of such regulators is shared by locicontaining NisT- and BcrAB-type transporters (Table 1 andsee below).

NisT-type transportersPhysiological role

Like the SunT-type transporters, the NisT-type systemsare responsible for the export of newly synthesized AMPs.However, because they lack a peptidase domain, thesubstrate peptides are translocated without cleavage andare subsequently processed by an extracellular serineprotease (Fig. 4B). Consistent with this, a gene encodinga protease was found in the genomic neighbourhood of67% of the analysed NisT-type transporters (Table 1). Theproteases are collectively referred to as LanP proteins,and cleavage of the leader peptide has been experimen-tally shown for, e.g. NisP (nisin) (Kuipers, 2004) or PepP(Pep5) (Meyer et al., 1995). Some substrates for NisT-type transporters, for example the four-peptide bacteri-ocin aureocin A70, are not cleaved (Netz et al., 2001).Export activity has been shown experimentally for someNisT-type proteins. For example Pep5 production wasreduced to 10% in the absence of PepT, and the AMP wasfound to accumulate intracellularly in this strain (Meyeret al., 1995). Deletion of NisT abolished secretion of nisin,while modified and thus active nisin was found to bepresent intracellularly, showing that modification wasindependent of transport (Qiao and Saris, 1996). Similarly,SpaT was shown to be required for subtilin export (Kleinet al., 1992) and AurT for aureocin A70 production (Netzet al., 2001). Low amounts of AMPs detected in theculture supernatants of such transporter mutants havebeen attributed to export by alternative, as yet unidentifiedtransport systems (Netz et al., 2001), but could also bedue to cell lysis caused by intracellular accumulation ofthe AMP, as was shown for subtilin (Klein et al., 1992).

Transport mechanism

Prior to export, lantibiotic AMPs are modified by dehydra-tion and thioether ring formation catalysed by LanB andLanC proteins respectively. For subtilin and nisin, theexistence of a multimeric complex between the modifica-tion and transport proteins, i.e. SpaBCT and NisBCT,respectively, was shown (Siegers et al., 1996; Kiesauet al., 1997). The arrangement of proteins in the formercomplex was suggested to be SpaC–SpaB–SpaT,because no contacts between SpaC and SpaT could bedetected (Kiesau et al., 1997). However, complex forma-tion was not found to be necessary for transport, becausein the absence of NisC, a NisBT complex could still dehy-drate and export pre-nisin that was lacking thioether rings.

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Furthermore, NisT alone could also export unmodifiedpre-nisin or even non-lantibiotic peptides, as long asthese carried the nisin leader sequence, showing that it isa rather broad-spectrum transporter and specificity isdetermined by the leader peptide (Kuipers, 2004). Theprotease NisP can only cleave the leader if the peptidecontains lanthionine residues, but cleavage is not coupledto transport, because externally added pre-nisin is alsocleaved by NisP (Kuipers, 2004). Thus, in contrast to theSunT-type transporters, transport and leader processingare separable processes in the biosynthesis of substratepeptides for NisT-type transporters.

Analysis of the genomic context of NisT-type transport-ers did not reveal any genes for the accessory proteinsthat were commonly found with SunT-type transporters(Table 1). It should be noted, however, that the data set forNisT-type transporters is small and likely not fully repre-sentative, because these systems could not be identifiedfrom sequence- or domain-based database searches andwere instead collected from known AMP biosynthetic loci.Often, these loci were cloned and sequenced withoutknowledge of the genetic neighbourhood, and thus rel-evant genes outside the sequenced regions could bemissed.

Interestingly, the two highly similar transporters GdmTand EpiT for the lantibiotics gallidermin and epidermin,respectively, were found to possess accessory proteins,named GdmH and EpiH (Peschel et al., 1997). BecauseEpiT contains a frameshift mutation, all experiments wereconducted with GdmT, and it was shown that this trans-porter requires the presence of either GdmH or EpiH forsubstrate export. These accessory proteins differ consid-erably from those described above for SunT-type trans-porters and contain six predicted transmembrane heliceswith a hydrophilic domain located between helices V and VI(Peschel et al., 1997). Their precise role in transport isunknown, and no corresponding genes were found in theloci of the remaining NisT-type transporters analysed here.

Regulation

Expression of NisT-type transporters is regulated togetherwith the other genes of the biosynthetic loci. About half ofthe loci analysed here contain a TCS consisting of atwo-transmembrane-helix histidine kinase with an extra-cellular sensory domain and an OmpR-like responseregulator (Table 1, Fig. 4B). This type of histidine kinaseshas been termed ‘prototypical periplasmic sensing’(Mascher et al., 2006). However, regulation of NisT-typetransporters by periplasmic sensing TCSs is only found inloci that also contain a LanFEG-type transporter, forexample the nisin biosynthesis locus that is regulated byNisRK (Kuipers et al., 1995), and is therefore discussed inthe following section. None of the NisT-type transporterswas found associated with a peptide quorum sensor TCS,although again the small size of the data set should bekept in mind.

As described for the SunT-type transporters, the NisT-type systems were also commonly found associated withan XRE family transcriptional regulator (Table 1, Fig. 4B).One example for this is LasX, which is required for theexpression of the lactocin S biosynthetic operon lasA–Wof Lactobacillus sakei (Skaugen et al., 2002). The signalleading to activation of las operon expression by LasX isnot known, but it does not appear to be autoinductionby lactocin S, because expression is unaltered in alasT mutant, which should be defective in AMP export(Skaugen et al., 2002). The biosynthetic locus for mutacinI in S. mutans is also positively regulated by an XRE-typeregulator, MutR, which is encoded upstream of the mutAoperon, and again the signal for MutR activation is notknown (Kreth et al., 2004). Mutacin I production is further-more subject to regulation via quorum sensing, resultingin AMP production during growth at high cell densities. Incontrast to the AMP Smb described above, this regulationdoes not occur via the ComCDE system, but instead viaautoinducer-2 (AI-2) signalling: the AI-2 synthase LuxS

Fig. 4. Summary of transport function and regulation for the five transporter groups. The cytoplasmic membrane is indicated as a horizontalbeige bar with the outside of the cell at the top. Transporters and associated resistance proteins are drawn in green, TCSs and otherregulators in blue using similar shades as in Fig. 2. AMPs are shown as black stars, leader peptides as wave shapes, transmembrane helicesas barrels, ATPase domains as ovals with the label ‘ATP‘ to indicate ATP hydrolysis, peptidase domains as ‘pacman’ symbols, the catalyticdomains of histidine kinases as rectangles, response regulators as diamonds, XRE regulators as labelled hexagons, genes as open arrowsand promoters as bent arrows. The positions of ATPase domains relative to permeases and of promoters relative to transporter genes waschosen arbitrarily; likely dimerization of permease subunits in (A), (B) and (D) is not shown for reasons of simplicity. AMP transport is indicatedby bold dashed arrows. Phosphotransfer within TCSs and gene activation are indicated by curved arrows, expression of transporter genes bydotted arrows. Unknown steps are labelled with a question mark. Where two different modes of gene regulation were found for a group oftransporters, both are shown below each other and marked by lower case Roman numerals.A. SunT group transporters.B. NisT group transporters. The peptidase is shown in yellow. Regulation by the shown TCS only occurs when the locus also contains aLanFEG-type transporter as indicated by genes in parentheses.C. LanFEG group transporters. LanI or LanH immunity proteins are shown as a red crescent.D. BceAB group transporters. Signalling between the transporter and the TCS is indicated by a double-headed arrow.E. BcrAB group transporters. UppP proteins are shown as a purple lozenge shape, undecaprenyl-(pyro)phosphate as a white lightning bold;the phosphorylation state is indicated. YydIJ-type transporters were not included in the diagram due to lack of information.See text for details and references.

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somehow represses transcription of irvA, which itselfencodes a transcriptional repressor of mutR and the mutAoperon, which inhibits mutacin I biosynthesis (Merrittet al., 2005).

Expression of the genes for gallidermin and epiderminexport, gdmT, gdmH, epiT and epiH, is positively regu-lated by the DNA-binding protein EpiQ that also regulatesthe remaining biosynthetic genes (Peschel et al., 1993;1997). EpiQ does not belong to the XRE family of tran-scriptional regulators, but instead contains a DNA-bindingdomain with similarity to OmpR-like response regulators(Peschel et al., 1993). How EpiQ is activated, for exampleif it requires phosphorylation by a histidine kinase(although a typical receiver domain is missing), or if it isacting as a stand-alone regulator is not known.

LanFEG-type transportersPhysiological role

Nearly all transporters of this type characterized to dateare involved in self-protection of lantibiotic producingstrains, and accordingly all recognize a lantibiotic sub-strate (Table 2). Interestingly, however, only about a thirdof LanFEG-type transporters included in the phylogeneticanalysis above were found to be associated with lantibi-otic biosynthesis genes (Table 1), suggesting that theirfunction is not restricted to self-protection. Furthermore,24% were neighboured by transposases or insertionsequences, indicating a high degree of genetic mobility(Table 1). As an example, the best-characterized biosyn-thetic locus, that of nisin, is located on a conjugativetransposon (Dodd et al., 1990). Consistently with theirrole in self-protection, most LanFEG transporters studiedto date have a very narrow substrate range, providingresistance only against the produced lantibiotic or struc-turally very similar peptides [e.g. gallidermin and epider-min but not nisin are transported by EpiFEG (Otto et al.,1998)]. In contrast, the CprABC transporter (for cationicAMP resistance) of Clostridium difficile, which is notassociated with any obvious biosynthesis genes, has abroader substrate range providing resistance against bothnisin and gallidermin (McBride and Sonenshein, 2010).Although more such stand-alone transporters will have tobe investigated to draw any general conclusions, it isconceivable that in systems not associated with biosyn-thetic loci a more relaxed specificity is advantageous forprotection against a range of lantibiotics.

Transport mechanism

The direction of substrate transport has been determinedfor several LanFEG-type transporters as a movement ofthe lantibiotic from the cytoplasmic membrane to the

culture supernatant, resulting in a shift of the distributionequilibrium between target-associated and free peptide(Otto et al., 1998; Stein et al., 2003; 2005; Okuda et al.,2008; 2010) (Fig. 4C). However, the question arises as tohow transport can be a resistance mechanism against cellwall-active AMPs at all: the cellular targets (UPP, lipid IIetc.) of these compounds are located on the surface of thecytoplasmic membrane. Thus, at first sight, the AMP willsimply re-associate with its target in a time limited only bythe rate of diffusion. For LanFEG-type transporters, theanswer may be found in the high co-occurrence (78%) withso-called immunity proteins (Table 1, Fig. 4C). Two typesof such proteins have been described. LanI-type proteinsare tethered to the membrane surface via an N-terminallipoprotein anchor, while LanH-type proteins contain threetransmembrane helices with the N-terminus locatedintracellularly. The high number of membrane-associatedhypothetical proteins found in the neighbourhood ofLanFEG-type transporters (Table 1) suggests that theco-occurrence of these transporters with immunity proteinshas even been underestimated. Both types of immunityproteins appear to function in concert with their LanFEG-type transporters as described for well-understoodexample systems below.

Self-protection against nisin in producer strains of Lac-tococcus lactis is mediated by the transporter NisFEG andthe LanI-type immunity protein NisI. Each system by itselfcan confer some level of resistance, yet full resistance isonly achieved in the presence of both (Stein et al., 2003).Similar results have been obtained for self-protection ofB. subtilis against subtilin by SpaFEG and SpaI (Steinet al., 2005). There is as yet no experimental evidence tounequivocally show whether full resistance is due to acooperative action of transporter and immunity protein orwhether both act independently. Stein and colleaguesreported that the resistance levels provided by NisFEG andNisI are additive, arguing for the latter mechanism (Steinet al., 2003). Another study showed that NisI is present asa membrane-anchored protein but additionally exists in alipid-free form that is also able to bind nisin (Takala et al.,2004). The lipid-free form mediated only a low level ofresistance in the absence of NisFEG, but this was signifi-cantly increased in the presence of the transporter, sug-gesting a cooperative mode of action (Takala et al., 2004).The authors propose a mechanism of cooperativity wherethe NisFEG transporter removes nisin from the membraneproducing a high local concentration of free nisin, which isthen bound by lipid-free NisI and moved away from the cellby diffusion (Takala et al., 2004).

Resistance of L. lactis against the two-peptide lantibi-otic lacticin 3147 is mediated by the transporter LtnFE(missing the second permease gene) and the immunityprotein LtnI (Draper et al., 2009). LtnFE or LtnI alone eachprovided some degree of protection, but together medi-

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ated higher resistance than the additive contribution ofeach system, again arguing for cooperativity (Draperet al., 2009). This effect was only observed for the two-peptide Ltna:Ltnb complex of lacticin 3147, whereas LntIand LtnFE acted independently against the individualLtna or Ltnb peptides (Draper et al., 2009). Interestingly,orthologues of both systems, e.g. an LtnI homologue fromB. licheniformis and an LtnFE homologue from Entero-coccus faecium could also impart lacticin 3147 immunityto L. lactis and also showed synergistic effects upon coex-pression, indicating that cooperativity is a general trait insuch systems (Draper et al., 2009).

Protection of S. warneri against nukacin ISK-1 is pro-vided by the NukFEG transporter and the LanH-typeimmunity protein NukH. Heterologous studies in L. lactisshowed that, as with the LanFEG–LanI systems, NukFEGand NukH together mediated higher levels of resistancethan the additive effect from expressing either determi-nant by itself (Aso et al., 2005). Individual expression ofNukFEG imparted a stronger resistance than expressionof NukH alone, showing that the transporter was the majorresistance determinant (Aso et al., 2005). A more detailedanalysis of the resistance mechanism showed thatL. lactis expressing NukH bound more nukacin than acontrol strain and that coexpression of NukFEGH resultedin an energy-dependent decrease in cell-associatednukacin (Okuda et al., 2008). This led the authors to thehypothesis that NukH captures nukacin and this NukH-bound substrate is then transported to the extracellularspace by NukFEG, implying that NukH functions as asubstrate-binding protein for the transporter (Okuda et al.,2008). Of note, for gene annotation and functional studiescare should be taken that these LanH proteins are notconfused with homologues of GdmH. The former havethree transmembrane helices and are functionally associ-ated with LanFEG-type transporters, while the latter havesix transmembrane helices and act as accessory proteinsfor NisT-type transporters as described above.

While most ABC transporters contain a so-called Q-loopmotif in their ATPase domain, Okuda and colleaguesreported that LanFEG-type transporters appear to possessa conserved glutamate (E) instead of the glutamine (Q)residue (Okuda et al., 2010). Mutation of this E-loop motifof the nukacin transporter NukFEG to the conventionalQ-loop however only had minor effects on immunity(Okuda et al., 2010). An analysis of the multiple sequencealignment of ATPases generated during the phylogeneticanalysis presented in Fig. 1A showed that indeed allLanFEG-type transporters contained a glutamate at theposition corresponding to position 85 in NukF. Further-more, all BcrAB-type transporters also contained thisresidue (position 84 in B. licheniformis BcrA), while theNisT-, SunT- and BceAB-type transporters possess theconventional Q-loop motif (not shown), again supporting

the common evolutionary origin of LanFEG and BcrABtransporters.

Regulation

Nearly three quarters of the LanFEG-type transportersanalysed here were found associated with a TCS contain-ing a prototypical periplasmic sensing histidine kinase(Table 1, Figs 2 and 4C). The best-characterized examplesfor such TCSs, NisRK and SpaRK regulating biosynthesisand immunity for nisin and subtilin, respectively, have beenpreviously reviewed in detail (Kleerebezem, 2004). There-fore I will here just give a brief summary on their function.

As with many lantibiotics, production of nisin A is autoin-duced in response to extracellular nisin A, nisin Z or nisinU (but not other lantibiotics) (Kuipers et al., 1995; deRuyter et al., 1996; Ra et al., 1996; Wirawan et al., 2006).This induction is highly sensitive, requiring only 30 pMnisin corresponding to about five molecules per cell andis triggered by nisin binding to the sensor kinase NisK(Kuipers et al., 1995). Interestingly, the observed cross-induction between different variants of nisin can occureven between L. lactis (nisin A producer) and Streptococ-cus uberis (nisin U producer), showing that AMPs canfunction in quorum sensing even between different bac-terial genera (Wirawan et al., 2006). Another example forinter-species communication is the cross-induction of sali-varicin A and A1 production by Streptococcus salivariusand S. pyogenes respectively (Upton et al., 2001). NisRKregulates nisin production from two promoters, PnisA (nisinproduction and nisI) and PnisF (nisFEG), whereas expres-sion of the nisRK structural genes is constitutive (deRuyter et al., 1996; Ra et al., 1996). Regulation of subtilinbiosynthesis by SpaRK is organized in a similar fashion,but expression of all immunity determinants, i.e. SpaI andSpaFEG, is regulated from a common promoter, PspaI

(Klein and Entian, 1994; Stein et al., 2002b).The mersacidin biosynthesis locus of Bacillus sp. HIL

Y-85 contains a dedicated TCS, MrsR2–MrsK2, that regu-lates expression of the immunity transporter, MrsFGE, butnot of mersacidin production (Guder et al., 2002). Instead,production (but not immunity) is positively controlled by theorphan response regulator MrsR1. It is not known whetherMrsR1 requires phosphorylation for activation and, if so,which is its cognate sensor kinase, but a potential role forMrsK2 in MrsR1 regulation was excluded (Guder et al.,2002).

An exception to this mode of regulation is the lacticin3147 self-resistance operon ltnRIFE. It is not controlled bya TCS but instead by the XRE family transcriptional regu-lator LtnR. Mutants in ltnR were found to be hyperresistant,while overexpression of the regulator caused increasedsensitivity to lacticin 3147, showing that LtnR acts as arepressor of ltnRIFE (McAuliffe et al., 2001). The lacticin

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3147 biosynthesis operon is transcribed divergently fromthe resistance genes and appears to be constitutivelyexpressed (McAuliffe et al., 2001).

BceAB-type transportersPhysiological role

BceAB-like transporters mediate resistance against AMPs(Table 2) but are not found associated with biosyntheticloci (Table 1). The only exception to this is the SalXYtransporter that is part of the salivaricin A biosynthesislocus (Upton et al., 2001). For most BceAB-type trans-porters several substrates have been identified, and oftenone transporter will recognize structurally very differentAMPs (summarized in Gebhard and Mascher, 2011). Sub-strates for BceAB-type transporters not only include lan-tibiotics, class II bacteriocins or bacitracin, but alsoglyocpeptides, b-lactam antibiotics and defensins andcathelicidins of mammalian origin (Table 2). In that regardBceAB transporters differ markedly from the other resist-ance transporters, which are more restricted in their sub-strate range and transport either bacitracin (BcrAB group)or lantibiotics (LanFEG group) (Table 2). The molecularmechanisms underlying this surprisingly broad substraterange of BceAB-type transporters are not understood, asdiscussed below.

Genomic context analyses show no significantco-occurrence of genes with one exception: the vastmajority of BceAB-type transporters are encoded next to aTCSs where the histidine kinases contain two transmem-brane helices without an extracellular domain, so-called‘intramembrane-sensing’ histidine kinases (Table 1,Fig. 4D) (Mascher, 2006; Dintner et al., 2011). As was firstdescribed for the eponymous bacitracin-resistance trans-porter BceAB of B. subtilis, these transporters are not onlyresponsible for resistance but are also required for signal-ling: the associated sensor kinase BceS was found to beunable to detect bacitracin in the absence of BceAB,which led to the proposition that the transporter containsthe actual sensory domain of the system (Bernard et al.,2007; Rietkötter et al., 2008). Since then, the same func-tional relationship between transporter and TCS has beenreported for a number of related modules (Ouyang et al.,2010; Hiron et al., 2011; Staron et al., 2011; Falord et al.,2012). Furthermore, BceAB-like transporters and BceRS-like TCSs were found to have co-evolved, supporting thetight functional link between these systems (Dintner et al.,2011).

Transport mechanism

In contrast to the previously described transporter groups,the direction of substrate translocation by BceAB-typetransporters is not yet known. It has been proposed that

these systems may act as importers, removing the AMPfrom its site of action (i.e. the cell surface) followed byintracellular inactivation through degradation (Rietkötteret al., 2008; Hiron et al., 2011). Yet it is equally possiblethat the transport mechanism is more similar to that ofLanFEG-type transporters, removing the AMP from thecell membrane to the extracellular space. However, nocommon association of BceAB-type transporters with anypotential auxiliary proteins such as the LanI/LanH proteinswas found (Table 1) (Dintner et al., 2011). The distinguish-ing feature of BceAB-type transporters is their large extra-cellular domain (ECD), and it could be speculated that thisdomain might play a role akin to that of LanI or LanHproteins, but to date no experimental data are available tosupport such a mode of action.

Substrate binding by BceAB-type transporters appearsto occur via their ECD, which was shown by a domain-swap experiment between two such transporters fromStaphylococcus aureus. A derivative of the colistin trans-porter VraFG that contained the ECD of the bacitracintransporter VraDE provided resistance against bacitracin,but no longer against its original substrate, colistin (Hironet al., 2011). Despite this, it is still unclear how the differentsubstrates are recognized and distinguished. As men-tioned above, most BceAB-type systems transport a rangeof AMPs, and the substrates for a single transporter candiffer considerably in their structure while at the same timeother structurally related compounds are not recognized.For example PsdAB of B. subtilis transports the lantibioticactagardine, but not the similar mersacidin. The sametransporter also recognizes one lipodepsipeptide, endura-cidin, but not another, ramoplanin (Staron et al., 2011).Further, while VraDE of S. aureus can detoxify both nisinand bacitracin, B. subtilis employs two separate transport-ers, PsdAB and BceAB, respectively, for resistance againstboth compounds (Ohki et al., 2003b; Hiron et al., 2011;Staron et al., 2011). An in-depth phylogenetic analysis ofBceAB-like transporters further showed that no correlationexisted between phylogenetic group and substrate range(Dintner et al., 2011).

Another interesting aspect of BceAB-type transporters istheir role in signal transduction. Again, the mechanism ofcommunication between the transporter and TCS is notknown, but parallel phylogenetic analyses (Dintner et al.,2011) and bacterial two-hybrid assays (Falord et al., 2012)suggest a direct interaction between the transport per-mease and histidine kinase. Importantly, the transportprocess itself is required for the signalling process,because mutations that prevent ATP hydrolysis and thussubstrate translocation abolish signal transduction(Rietkötter et al., 2008). This requirement for ATP hydroly-sis even holds true for the dedicated sensing transportersdescribed below, implying that they must be capable oftransport, even though they do not mediate resistance

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(Hiron et al., 2011). It therefore appears that the ability totransport an AMP does not necessarily equate to providingresistance. An explanation for this may be that the trans-port rate of the sensing transporters is too low to counteractthe inhibitory effects of the AMP while it is still sufficient forsignalling, but experimental evidence for this is missing.

Regulation

All BceAB-type transporters studied to date are regulatedby a TCS that is most commonly encoded in an adjacentoperon (Fig. 2) (Joseph et al., 2002; Dintner et al., 2011).The histidine kinases of these systems belong to the groupof intramembrane-sensing kinases (Mascher, 2006;Mascher et al., 2006), and the response regulators to theOmpR group (Fig. 4D). The consensus binding sequencefor the response regulators associated with BceAB-typetransporters has been identified as TNACA-N4-TGTAAwithanAT-rich central 4 nt spacer (de Been et al., 2008; Dintneret al., 2011). Expression of the transporter is induced in thepresence of sublethal concentrations of its substrate AMP,and this requires the presence of both the TCS and thetransporter itself, as described above. In some cases, adedicated sensing transporter is present to aid in inductionof a second transporter that in turn mediates the actualresistance. Such a scenario is for example found inS. aureus, where the TCS BraRS (also called NsaRS orBceRS) together with its sensing transporter BraDE (alsocalled NsaAB or BceAB) induces expression of the trans-porter VraDE that provides resistance against bacitracin ornisin (Pietiäinen et al., 2009; Hiron et al., 2011; Kolar et al.,2011; Yoshida et al., 2011). Additionally, S. aureus con-tains a third BceAB-like transporter, VraFG, that acts as asensor for the TCS GraRS (also termed ApsRS) (Li et al.,2007a; Meehl et al., 2007; Falord et al., 2011; 2012). TheTCS GraRS is itself unusual in that it requires an accessoryprotein, GraX, in order to induce gene expression and hastherefore been termed a three-component system (Liet al., 2007b; Falord et al., 2012). The exact role of GraX isnot understood and no other intramembrane-sensing TCSstudied to date possesses such an accessory protein.

Many Firmicutes bacteria contain several, in somecases up to six paralogues of BceAB-like transporters(Dintner et al., 2011). Their respective roles in resistanceand regulatory interplay have been studied in most detailfor B. subtilis and S. aureus, and one striking differencebetween the two organisms is the degree of cross-regulation among the paralogous modules. The threesystems of B. subtilis (BceRS-AB, PsdRS-AB and YxdJK-LM) are well insulated from each other, with only a minordegree of cross-phosphorylation observed from the histi-dine kinase BceS to the response regulator PsdR (Rietköt-ter et al., 2008). In contrast, in S. aureus the transporterVraDE, which does not possess its own TCS, is regulated

by the TCS BraRS and the sensing transporter BraDE, andaccording to some studies also by the TCS GraRS and itssensing transporter VraFG (Li et al., 2007a; Gebhard andMascher, 2011; Hiron et al., 2011). This difference in regu-latory complexity between B. subtilis and S. aureus is alsoreflected in the respective regulons of the involved TCSs.In B. subtilis the associated BceAB-type transporter is thesole target of regulation (Ohki et al., 2003b; Rietkötteret al., 2008; Staron et al., 2011), while GraRS and BraRSof S. aureus additionally regulate genes of the cell wallstress response, including mprF, the dtlABCD operon(GraRS) and genes for cell wall biosynthesis (BraRS)(Kolar et al., 2011; Falord et al., 2012). Induction of theseadditional genes will doubtlessly contribute to resistanceagainst cell wall-active antibiotics, and it therefore remainsto be determined which of the broad range of inducingsubstrates, particularly of the three staphylococcalsystems (Gebhard and Mascher, 2011), are actuallydetoxified by the transporter itself or simply counteractedby the general changes in the cell envelope.

A similar extended regulon might also exist inS. mutans, because in addition to the transporter MbrAB(also termed BceAB) three hypothetical proteins wereidentified as potential targets for the TCS MbrCD (alsotermed BceRS) from an in silico search for the responseregulator binding consensus (Ouyang et al., 2010). In Lis-teria monocytogenes, the TCS VirRS regulates expres-sion of a BceAB-like transporter, AnrAB, as well as of thedlt operon and mprF (Mandin et al., 2005; Collins et al.,2010). The sensing transporter for VirRS has not yet beenidentified, but a likely candidate is the uncharacterizedBceAB-like transporter Lmo1747-1746, which is encodedin the same locus as VirRS (Mandin et al., 2005; Dintneret al., 2011). An explanation for the much simpler regula-tion in B. subtilis may be the presence of several extra-cytoplasmic function (ECF) sigma factors, e.g. SigW orSigM, which regulate the more general cell wall stressgenes like dlt and mprF (Mascher et al., 2007). S. aureus,S. mutans and L. monocytogenes all possess no or only asingle ECF sigma factor (Jordan et al., 2008), and accord-ingly the TCSs associated with BceAB-like transportersmay have evolved to control a broader regulon.

BcrAB- and YydIJ-type transportersPhysiological role

BcrAB-type transporters are also involved in resistanceagainst AMPs, but they act specifically against the cyclicpeptide bacitracin. The eponymous system is found in thebacitracin producer B. licheniformis ATCC10716, where itis encoded in the biosynthesis locus bacABC–bacRS–bcrABC and confers self-resistance (Podlesek et al.,1995; Neumüller et al., 2001). A second characterized

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example is BcrAB from Enterococcus faecalis AR01/DGVS, a clinical isolate that displayed high-level baci-tracin resistance (Manson et al., 2004).

Both transporters are co-transcribed with genes encod-ing putative undecaprenyl-pyrophosphatases (UppP),bcrC in B. licheniformis and bcrD in E. faecalis. UppPs canmediate bacitracin resistance by dephosphorylation of thecellular target of the AMP, undecaprenyl-pyrophosphate(UPP). While a BcrC orthologue was indeed shown toconfer resistance to B. subtilis (Cao and Helmann, 2002;Mascher et al., 2003; Ohki et al., 2003a), no function ofBcrD in E. faecalis has been identified to date (Mansonet al., 2004; Gauntlett et al., 2008).Additionally, both trans-porter operons are preceded by genes for transcriptionalregulators, bacRS in B. licheniformis and bcrR in E. faeca-lis, which are discussed in detail below. Overall, analysis ofthe genomic context of these transporters showed thattheir loci are small and restricted to the transporter itself,often with genes for a regulatory system and a UppP(Table 1, Fig. 2), supporting their involvement in resistanceonly. Among environmental isolates of enterococci, bcrABgenes appear widely distributed (Matos et al., 2009), whichsuggests genetic mobility of the encoding regions. Indeed,efficient transfer of the bcr genes from the clinical E. fae-calis isolate AR01/DGVS to the bacitracin-sensitive labo-ratory strain JH2-2 has been demonstrated (Manson et al.,2004).

The YydIJ-like transporters possess the same domainarchitecture as the BcrAB transporters, but form a distinctbranch in the phylogenetic tree (Fig. 1). Only one examplehas been characterized to date, YydIJ of B. subtilis(Butcher et al., 2007). This transporter is encoded as partof the yydFGHIJ locus, which additionally contains genesfor a small peptide (YydF) and its modification (YydG) andprocessing (YydH). YydF most likely has a cell envelope-disturbing effect, because it induces the LiaRS-mediatedcell wall stress response in B. subtilis (Butcher et al.,2007). YydIJ may act as an exporter of YydF or alterna-tively in YydF self-resistance, as discussed below. Thedistribution of YydIJ-like transporters among Firmicutesappears to be rather restricted. The complete yydF–Jlocus was only found in some strains of B. subtilis, whileBacillus cereus possessed the transporter neighbouredby a truncated yydH gene and several genes for trans-posases and an integrase. Other loci contained only thetransporter (Table 1, Fig. 2). As with the BcrAB-type trans-porters, acquisition of yyd loci by horizontal gene transferhas been discussed (Butcher et al., 2007).

Transport mechanism

To date, no information is available on the mechanism ofAMP transport by BcrAB-type transporters. As mentionedpreviously, for BceAB-type transporters the possibility of

AMP import followed by enzymatic inactivation inside thecell has been proposed (Rietkötter et al., 2008; Hironet al., 2011). Experimental evidence, however, is availableonly for the LanFEG-type transporters, which removetheir lantibiotic substrates from the cytoplasmic mem-brane and release it to the supernatant as describedabove (Otto et al., 1998; Okuda et al., 2008; 2010). Thephylogenetic tree of AMP transporters (Fig. 1) shows thatthe BcrAB-type transporters are ancestral to the LanFEGtransporters, thus a similar transport mechanism in bothgroups should be assumed.

Some indirect evidence for such a ‘hydrophobic vacuumcleaner’ mechanism (Chang, 2003) of BcrAB-type trans-porters may be found in the transcriptional response ofthe bcrR–bcrABD locus of E. faecalis to bacitracin. Inthe presence of the BcrAB transporter, the sensitivity ofbacitracin detection by the regulator, BcrR, is greatlydecreased (Gauntlett et al., 2008). These data were inter-preted as removal of bacitracin by BcrAB from the site ofdetection by BcrR (Gauntlett et al., 2008), which is mostlikely at or within the cytoplasmic membrane, becauseBcrR has no extracellular domains (Gebhard et al., 2009).Similarly, cells of B. subtilis lacking YydIJ were found todisplay a stronger YydF-induced Lia response comparedwith YydIJ-positive strains (Butcher et al., 2007). Theauthors discussed this as caused by accumulation of theYydF peptide inside the cell and therefore propose YydIJ toact in peptide export (Butcher et al., 2007). In light of thedata available now for LanFEG- and BcrAB-type transport-ers, an alternative explanation could be that YydIJ confersself-resistance by removing YydF from the membrane, andthus deletion of the transporter might increase the cell wallstress detected by the Lia system. Such a mechanismwould also be consistent with the finding that cells lackingYydIJ can still induce the Lia response in a neighbouringyyd-deleted strain (Butcher et al., 2007).

Still the question remains how the removal of an AMPfrom the cell membrane to the culture supernatant canconfer resistance. For the bacitracin-resistance transport-ers, the actual resistance mechanism may be tightly linkedto the activity of UppPs. As shown in Table 1, 77% of theanalysed BcrAB-type transporters are associated withUppP-encoding genes located downstream of the trans-porter (Fig. 2), and it is therefore likely that all three genesform an operon. Upon induction of this operon, the shift inthe distribution equilibrium between cell-associated andfree bacitracin mediated by the transporter may be suffi-cient to enable rapid dephosphorylation of UPP by theUppP (Fig. 4E). Because bacitracin has poor affinity formonophosphates (Storm and Strominger, 1973), resist-ance is thus ensured. This model is supported by an earlierreport that all three genes (bcrABC) of B. licheniformiswere necessary to confer bacitracin resistance to theheterologous host B. subtilis, which led to the original

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assumption that BcrC was a third component of the trans-porter (Podlesek et al., 1995). In E. faecalis the UppP BcrDwas shown to be dispensable for bacitracin resistance, butit could not be ruled out that a second paralogue wasencoded elsewhere on the genome (Gauntlett et al.,2008).

Regulation

The majority of BcrAB transporter encoding loci analysedhere contained the same type of TCS as found with BceAB-like transporters, i.e. possessing an intramembrane-sensing histidine kinase and an OmpR family responseregulator (Figs 2 and 4E, Table 1). For the bacitracin bio-synthesis locus from B. licheniformis, the TCS BacRS wasshown to regulate expression of bcrABC, but not of baci-tracin biosynthesis itself (Neumüller et al., 2001). Experi-mental data suggest that BacR acts as a negativeregulator, repressing transcription of bcrABC in theabsence of bacitracin (Neumüller et al., 2001), which isin contrast to other Bce-like TCSs studied to date thatfunction by positive regulation (Ohki et al., 2003b; Liet al., 2007b; Collins et al., 2010; Hiron et al., 2011). Itis not known how bacitracin is sensed by BacS. Theintramembrane-sensing histidine kinases associated withBceAB-type transporters are known to strictly depend ontheir corresponding transporter for stimulus detection(Rietkötter et al., 2008; Gebhard and Mascher, 2011; Hironet al., 2011), and it is therefore questionable whether thestructurally very similar BacS is able to perceive a stimulusdirectly. While it is possible that BcrAB-type transportersmay also be involved in the signalling process, it has beenshown for E. faecalis BcrAB that it is not required forsensing (Gauntlett et al., 2008). It should however benoted that this particular transporter is not regulated by aTCS.

One-third of the BcrAB-type transporters were found tobe associated with transcriptional regulators of the XREfamily, often containing four predicted transmembranehelices in their C-terminal domain (Fig. 4E). One examplefor this, BcrR of E. faecalis, has been studied in detail,and it was shown that it is indeed a membrane-boundDNA-binding protein (Gauntlett et al., 2008). BcrR bindsbacitracin directly, most likely at or within the cytoplasmicmembrane as mentioned above and subsequentlyinduces transcription from a single promoter upstream ofthe bcrABD operon (Gauntlett et al., 2008; Gebhard et al.,2009). The target promoter contains two inverted repeatsequences that are both necessary for induction, and it istherefore thought that it is bound by two dimers of BcrR.DNA binding was shown to be independent of bacitracin,suggesting that the promoter is constitutively occupied byBcrR, which may be important for a membrane protein inorder to find its target sequence (Gebhard et al., 2009). It

has been proposed that binding of bacitracin by BcrRleads to a change in the interaction between individualBcrR monomers or between BcrR and RNA polymerase(Gebhard et al., 2009), but the exact mechanism for tran-scription activation remains to be elucidated.

The yydIJ operon of B. subtilis is neighboured by aconvergently transcribed gene, yydK, which encodes aputative GntR-type regulator (Fig. 2); however, its functionis unknown.

Concluding remarksThis review to my knowledge presents the first study witha focus on the different types of AMP transporters found inFirmicutes bacteria without taking its primary motivationfrom AMP biosynthesis or resistance. Importantly, thegenomic context analysis showed that even SunT-typeand LanFEG-type transporters, which are normally con-sidered tightly linked to lantibiotic production, are notalways associated with biosynthetic genes (Table 1). Tofacilitate an independent comparison of the AMP trans-porters themselves, a new classification system based ondomain architecture was developed. ABC transporters areoften categorized according to the TCDB established bythe Saier group and available online (http://www.tcdb.org)(Saier et al., 2009). While this database also includesthe AMP transporters reviewed here, their classificationappears in parts somewhat inconsistent and only very fewexamples are included (two to seven per family). TheTCDB classification matches the one proposed here forthe LanFEG-type transporters (TCDB Peptide-5 Export-ers) and the BceAB-type transporters (TCDB Peptide-7Exporters). However, SunT-type transporters are found inthe TCDB families Peptide-1 Exporters and Peptide-2Exporters, while the NisT group is found in the Peptide-1Exporters and Peptide-4 Exporters families. The domain-based classification presented here appears more mean-ingful, because it accurately reflects the phylogeny of thetransporter groups, for example showing that the BcrAB-type transporters are ancestral to the LanFEG group(Fig. 1). Furthermore, genomic context analyses and areview of the available literature based on this new clas-sification revealed distinguishing as well as common char-acteristics regarding transport mechanism and regulation,which can be attributed to each group of transporters.

Accessory proteins

One striking observation is that the majority of AMP trans-porters from Firmicutes appear to function together withaccessory or auxiliary proteins. This is most obvious for theSunT group, where nearly half of the transporters analysedpossessed a dedicated accessory protein, and functionalevidence was available on their requirement for peptide

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export. While the genomic neighbourhood of the analysedNisT-type transporters did not appear to contain homolo-gous proteins, the example of the gallidermin transporterGdmT, which depends upon the unrelated accessoryprotein GdmH for transport, shows that at least someNisT-type transporters cannot function by themselves. Thisis perhaps not too surprising, given that SunT- and NisT-type systems form a common evolutionary group in thephylogenetic tree of AMP transporters (Fig. 1). It will beinteresting to see whether more GdmH-like proteins arefound as more NisT-like transporters are identified. Amongthe resistance transporters, no additional proteins aredirectly necessary for the transport process, but the highdegree of co-occurrence of LanFEG-type transporters withLanI or LanH immunity proteins, and of BcrAB-type trans-porters with UppP-encoding genes nevertheless suggestsa tight functional link (Fig. 4C and E). As discussed above,these proteins may be an important facet of the immunitymechanism: if the transporter’s role is to remove cellmembrane-associated AMPs and release them to theculture supernatant, a second protein may be needed toensure the AMP cannot re-associate with the cell, either bybinding it (LanI or LanH proteins) or by removing its cellulartarget (UppP proteins). The lack of association of BceAB-type transporters with any additional genes may hint at adiffering transport mechanism. Alternatively such a func-tion might be implied for their large extracellular domain,but as no experimental evidence is available to date, thisremains speculation.

Association with distinct regulatory systems

Genomic context analysis showed that each group oftransporters was found to be associated with a distincttype of regulatory system (Table 1, Fig. 4). SunT-typetransporters are commonly regulated by TCSs with apeptide quorum sensor kinase and LytR family responseregulator; LanFEG-type transporters are regulated byTCSs with a prototypical periplasmic sensing kinase andan OmpR family response regulator; BcrAB and BceAB-type transporters are regulated by TCSs with anintramembrane-sensing histidine kinase and again anOmpR family response regulator. NisT-type transporterswere often found associated with the same type of TCS asthe LanFEG transporters, but this is most likely due to thefact that these genetic loci in all cases also contained aLanFEG-type transporter (Table 1). Where no TCS wasfound in the neighbourhood of a transporter, the mostcommon regulatory genes encoded XRE family transcrip-tional regulators (Table 1, Fig. 4). Such regulators werefound with transporters of the SunT, NisT, BcrAB andLanFEG groups, and in several cases it has been shownexperimentally that they indeed regulate expression oftheir respective transporter.

Parallels to quorum sensing

All three types of TCSs as well as the membrane-boundXRE regulators are activated in the presence of the trans-porter’s substrate AMP, with the exception of some SunT-associated systems that are induced in response to adedicated pheromone. Particularly in the context of bio-synthesis loci for AMPs this mode of regulation is highlysimilar to quorum sensing mechanisms, and such con-nections between AMP production and cell–cell commu-nication have been drawn repeatedly (Upton et al., 2001;Kleerebezem, 2004; Wirawan et al., 2006). The questionthat is usually asked next is whether AMPs have primarilyevolved as a means for inter-species warfare or rather asa means for communication within bacterial populations.The strong correlation between the type of transporterand its associated regulatory system described here mayopen up new and promising avenues for investigationsaimed at finding the answer.

Open questions

Despite the considerable knowledge gained on AMPtransporters to date, several questions remain to beanswered. One central point is the direction of substratetransport by the BcrAB- and BceAB-type resistance trans-porters. Do they function the same way as LanFEG trans-porters, and if so, do the associated UppPs or other as yetunidentified proteins contribute to providing maximalresistance? A second important question concerns thesite of AMP binding, not only by the transporters but alsoby the sensory proteins. For BceAB-type transporters, theECD is the most likely candidate region, but where dotransporters without any obvious ligand-binding domains,i.e. LanFEG- and BcrAB-type systems, bind their sub-strates? The same can be asked of regulatory proteinssuch as the membrane-bound XRE regulators or theintramembrane-sensing histidine kinases associated withBcrAB-like transporters. And finally, what forms the basisfor substrate specificity? This question was posed abovefor the ECD of BceAB-type transporters, but also no infor-mation is available for example on how the periplasmicsensing histidine kinases found with LanFEG-type trans-porters distinguish between the different AMPs to providespecificity in signalling. No doubt future research will beaimed at shedding light on these aspects and continue todeepen our understanding on AMP transport in Firmicutesbacteria.

AcknowledgementsThe author would like to thank Thorsten Mascher for stimu-lating discussions before and during preparation of thisreview and for critical reading of the manuscript. I also extend

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my apologies to the authors of the numerous publications onAMP transporters that were not cited due to space restric-tions. Work in the author’s lab is supported by grants from theDeutsche Forschungsgesellschaft (GE2164/3-1) and theFonds der Chemischen Industrie.

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