The polypeptide core of Microcin E492 stably associates with the mannose permease and interferes with mannose metabolism

(1)

Article

Reference

The polypeptide core of Microcin E492 stably associates with the mannose permease and interferes with mannose metabolism

BIELER, Sylvain, SILVA, Filo, BELIN, Dominique

Abstract

Microcin E492 (MccE492) is an antibacterial protein whose activity on target cells requires ManYZ, the inner membrane component of the mannose permease. We show here that MceA, the polypeptide core of MccE492, stably associates with ManYZ both in the presence and in the absence of MceB, the MccE492 immunity protein. The two known physiological activities of the mannose permease were assayed in cells co-expressing MceA and MceB.

Under these conditions, growth on mannose as the sole carbon source is prevented; this was not observed in cells expressing only MceB. In contrast, susceptibility to bacteriophage λ infection was not affected.

BIELER, Sylvain, SILVA, Filo, BELIN, Dominique. The polypeptide core of Microcin E492 stably associates with the mannose permease and interferes with mannose metabolism. Research in Microbiology , 2010, vol. 161, no. 8, p. 706-10

PMID : 20674740

DOI : 10.1016/j.resmic.2010.07.003

Available at:

http://archive-ouverte.unige.ch/unige:25085

Disclaimer: layout of this document may differ from the published version.

1 / 1

(2)

The polypeptide core of Microcin E492 stably associates with the mannose permease and interferes with mannose metabolism

Sylvain Bie´ler, Filo Silva, Dominique Belin *

Department of Pathology and Immunology, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland Received 21 April 2010; accepted 15 July 2010

Available online 30 July 2010

Abstract

Microcin E492 (MccE492) is an antibacterial protein whose activity on target cells requires ManYZ, the inner membrane component of the mannose permease. We show here that MceA, the polypeptide core of MccE492, stably associates with ManYZ both in the presence and in the absence of MceB, the MccE492 immunity protein. The two known physiological activities of the mannose permease were assayed in cells co- expressing MceA and MceB. Under these conditions, growth on mannose as the sole carbon source is prevented; this was not observed in cells expressing only MceB. In contrast, susceptibility to bacteriophagelinfection was not affected.

Keywords:MceA; Bacteriocin; Immunity; Mannose; Bacteriophagel

1. Introduction

Bacteriocins are proteinaceous antibiotics produced by bacteria. Microcin E492 (MccE492) is secreted by Klebsiella pneumoniae RYC492 and active against various Enter- obacteriaceae (de Lorenzo, 1984; de Lorenzo et al., 1984;

Thomas et al., 2004). It is composed of a polypeptide core (MceA; Swiss-Prot Q9Z4N4, aa 16-99) with a C-terminal cova- lent modification (Thomas et al., 2004). MccE492 is believed to form pores in the inner membrane, leading to lethal disruption of membrane potential and integrity (de Lorenzo and Pugsley, 1985;

Destoumieux-Garzon et al., 2003). Bacteria producing MccE492 synthesize an immunity protein (MceB), which is also localized in the inner membrane (Lagos et al., 1999).

MccE492 bactericidal activity is strictly dependent on the inner membrane component of the mannose permease (Bie´ler et al., 2006). This permease is composed of two membrane proteins (ManY and ManZ) and one cytoplasmic protein (ManX) that are involved in the uptake of mannose and related

hexoses (Elliott and Arber, 1978; Erni et al., 1987; Williams et al., 1986). The mannose permease is also required for bacteriophagelDNA penetration across the inner membrane (Elliott and Arber, 1978; Erni et al., 1987).

The expression of MceA, the polypeptide core of MccE492, is toxic in itself. This activity is enhanced by targeting to the inner membrane, for instance, with a mutant PhoA signal sequence (PhoA73) (Bie´ler et al., 2006). Bacteria that express the immunity protein MceB or that do not synthesize ManYZ are insensitive to MccE492, supplied extracellularly, and to both MceA and PhoA73-MceA, which exert their effect from the cytoplasm (Bie´ler et al., 2006). It is not yet known whether MceB acts by preventing MceA interaction with the mannose permease or by interfering at a later stage with the formation of toxic pores. In the latter case, MceB could, with or without MceA, affect the known functions of the mannose permease.

2. Materials and methods

2.1. Bacterial strains and plasmids

Escherichia coli strains AB3 (DmanXYZ::cat) and AB4 (manY::Tn10kan) are derivatives of DB503 (Bie´ler et al.,

* Corresponding author. Tel.:þ41 22 379 57 69; fax:þ41 22 379 57 46.

E-mail addresses:sylvain.bieler@gmail.com(S. Bie´ler),Maria-Filomena.

Silva@unige.ch(F. Silva),dominique.belin@unige.ch(D. Belin).

doi:10.1016/j.resmic.2010.07.003

(3)

2006). pBAD33 and pBAD101 have been described (Bie´ler et al., 2006; Guzman et al., 1995). pAB11 expresses PhoA73-MceA fusion under the control of the arabinose P_BAD promoter (Bie´ler et al., 2006). pAB23 expresses only MceB under the control of the P_BAD promoter of pBAD33 and is derived from pAB6 (Bie´ler et al., 2006). pJFLPM expresses ManX, ManY and untagged ManZ under the control of Ptac;

pJFP-H6M expresses ManY and hexahistidine (6H)-tagged ManZ (Esquinas-Rychen and Erni, 2001).

2.2. Membrane preparation

Cell fractionation was performed as described (Oliver and Beckwith, 1982), with the following modifications: cells were grown at 37C to anA_{600 nm}of 0.6 in 20 ml of LB and induced as indicated. Cells washed twice in 10 mM Tris pH 8 were resuspended in 2 ml of 20% sucrose, 30 mM Tris pH 8.

After addition of 200ml of lysozyme (0.5 mg/ml in 50 mM EDTA), cells were incubated for 15 min on ice. After addition of 40ml of 1 M MgCl₂, the spheroplasts were centrifuged for 10 min at 1500g at 4C, resuspended in 2 ml of 10 mM Tris pH 8, 0.1 M NaCl, 2 mM MgCl₂, 1 mM CaCl₂, 8 U/ml DNAse (Promega), frozen and thawed thrice, and finally centrifuged at 100,000gfor 1 h at 4C. Membrane pellets were resuspended in 100ml of 50 mM Tris pH 8, 10 mM EDTA, 0.1 M NaCl.

2.3. Purification and detection of hexahistidine (6H)- tagged protein complexes

Membranes (20ml) were solubilized in DDM buffer (20 mM Tris pH 8.1, 0.3 M NaCl, 10% glycerol, 1%

n-dodecyl-b-D-maltoside (DDM), 2 mM MgCl₂, 10 mM imidazole and 3.4 mg/ml E. coli polar lipids (Avanti Polar Lipids, Inc.)) or in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) supplemented with 2 mM MgCl₂at 4C for 1 h with agitation and centrifuged for 15 min at 13,000g at 4C. Supernatants were incubated at 4C for 30 min with 20ml of Ni-NTA agarose (Qiagen) and washed thrice with 1 ml of DDM buffer containing 30 mM imidazole and 1.5 mg/mlE. coli lipids or RIPA buffer. Pellets were resuspended in 40ml of SBX (80 mM Tris pH 6.8, 1%b-mercaptoethanol, 2% SDS, 0.01%

bromophenol blue, 10% glycerol, 10 mM EDTA), incubated for 20 min at 37C and eluates were loaded onto 16% SDS- polyacrylamide gels. MceA immunoblotting was performed as described (Bie´ler et al., 2006).

3. Results and discussion

3.1. MceA stably associates with ManYZ

A link between the activity of MccE492 and the presence of ManYZ had been established genetically (Bie´ler et al., 2006).

Fig. 1shows that PhoA73-MceA co-purified with 6H-tagged ManZ, while no PhoA73-MceA was recovered with cells expressing wild-type, untagged ManZ, from the pJFLPM

plasmid. Control experiments showed that SecY, an inner membrane protein that only interacts transiently with ManY and ManZ during their insertion in the membrane, was not detectably associated with 6H-tagged or untagged ManZ (Fig. 1A). This association is remarkably stable, since it is not only resistant to mild detergent (DDM buffer, Fig. 1A), but also to stronger detergents (RIPA buffer, Fig. 1B). PhoA73- MceA and ManZ may associate either through a direct interaction or through an indirect one involving, for example, ManY. Indeed, ManY and ManZ form a stable complex in the inner membrane (B. Erni, personal communication). These biochemical results indicate that a stable physical interaction can be detected between the mannose permease and the polypeptide core of MccE492.

Fig. 1. Binding of PhoA73-MceA to ManYZ in the absence or in the presence of MceB. Cultures of AB4 pJFLPM (ManYZ) or AB4 pJFP-H6M (ManY 6H- ManZ) harboring pAB11 (PhoA73-MceA) (þ) or the pBAD101 vector (), and pAB23 (MceB) (þ) or the pBAD33 vector (), were induced for 10 min with 0.2% arabinose and 0.5 mM IPTG and fractionated. (A) Membranes were analyzed by MceA immunoblotting either directly (top, 5ml of membranes) or after solubilization in DDM buffer and purification of 6H-tagged protein complexes (bottom, 20ml of membranes). The blot was reprobed with an anti- SecY antibody. (B) Membranes were solubilized in RIPA buffer.

707 S. Bie´ler et al. / Research in Microbiology 161 (2010) 706e710

(4)

3.2. MceB does not prevent the association of MceA with ManYZ

The association of PhoA73-MceA with ManYZ is not prevented by the expression of the immunity protein MceB (Fig. 1). Thus, MceB does not exert its protective action by blocking PhoA73-MceA membrane insertion or by preventing PhoA73-MceA from interacting with ManYZ (Fig. 2, model 2a). Therefore, MceB is most likely to be active at a later stage, for instance, by interfering with the toxic structure composed of both MceA and ManYZ. Since MceB synthesis abolishes PhoA73-MceA toxicity, higher amounts of PhoA73- MceA are produced in cells expressing MceB.

Interestingly, lactococcin A, a membrane-permeabilizing bacteriocin from the Gram-positive bacterium Lactococcus

lactis, is also dependent on the membrane components of the mannose permease for bactericidal activity (Diep et al., 2007).

Lactococcin A forms a stable complex with a tagged version of its cognate immunity protein and the mannose permease, but the immunity protein does not bind to the mannose permease in the absence of bacteriocin. Similar results were obtained with several other bacteriocins, including Listeria- active ones, which do not share sequence homology with MceA or lactococcin A (Diep et al., 2007).

3.3. Co-expression of MceA and MceB interferes with mannose metabolism

Since MceA associates with ManYZ, MceA expression could affect the two other known functions of the mannose permease (Fig. 2, model 2b). We tested these possibilities by measuring the capacity of cells stably expressing PhoA73- MceA and MceB to use mannose as a carbon source (Man^þ or Man phenotypes) and to be infected by bacteriophagel (l^S or l^R phenotypes). Strikingly, mannose metabolism is blocked when the expression of PhoA73-MceA and MceB is induced (Man phenotype), as evidenced by strong cell growth inhibition on minimal-mannose medium (Fig. 3A), while growth is normal on minimal-glycerol medium (Fig. 3B). Similarly, no mannose fermentation was detected on McConkey indicator plates upon PhoA73-MceA and MceB co-expression (data not shown). In contrast, expression of MceB alone had no visible effect on mannose metabolism. Since expression of PhoA73-MceA without MceB is lethal, its effect on mannose metabolism cannot be assessed.

The inhibition of growth observed upon induction of MceA and MceB was quantified in liquid cultures. In minimal- mannose medium, the doubling time increased by more than 7-fold upon induction; in contrast, growth in minimal glucose medium was only slightly decreased upon induction (Fig. 3C).

This probably reflects the fact that most of the glucose used under our culture conditions is imported by the PtsG system.

The situation is strikingly different in Lactococcus cells expressing the immunity protein and exposed to extracellular lactococcin A, since growth in the presence of mannose was barely affected (Diep et al., 2007).

Finally, growth in liquid cultures led us to determine that growth inhibition provoked by induction of MceA and MceB is reversible, since growth immediately resumes upon addition of glucose to induced cells (Fig. 3D).

In contrast, sensitivity to bacteriophage lvir infection, which makes clear plaques with sharp edges, is not signifi- cantly altered by PhoA73-MceA and MceB co-expression (l^S phenotype; data not shown). Thus, distinct portions of the mannose permease are likely to be involved in MccE492 sensitivity and bacteriophage l DNA penetration (Fig. 2, model 2b). Since l infection requires much less ManYZ activity than growth on mannose, a difference in the kinetics of plaque formation may not be visible, but the plaques were neither small nor minute. Although ManYZ inhibition by MceA and MceB has a measurable effect on mannose

Fig. 2. Models of MceA interaction with ManYZ and of MceB-mediated immunity. The thick arrows indicate possibilities documented by our own work. (1) In the absence of MceB, MceA interacts with ManYZ to induce membrane depolarization and cell death (Bie´ler et al., 2006; de Lorenzo and Pugsley, 1985). Since MccE492 can form cation-specific channels by itself in lipid bilayers (Lagos et al., 1993), the lethal complex may or may not contain ManYZ. (2) Possible mechanisms of immunity: A) MceB interferes with MceA-ManYZ association; B) MceA, MceB and ManYZ form a complex. This association may or may not affect the two known functions of the permease, mannose uptake and bacteriophage l DNA penetration.

Although we have not directly assessed the presence of MceB, similar experiments with aLactococcusbacteriocin system demonstrate the association of the tagged immunity protein in the complex (Diep et al., 2007). For ManYZ, the inner circle represents the portion of the complex used for DNA penetration and the outer circle that used for mannose import. We show here thatman^þcells expressing MceA and MceB have a Manl^Sphenotype (thick arrow and box).

(5)

transport, it may not prevent the occasional penetration of a single bacteriophage DNA molecule.

Although it is not known whether bacteriophage DNA penetration and mannose uptake involve the same structural element, the diameter of a double-stranded DNA is signifi- cantly larger than that of mannose, a monosaccharide. Thus, the transfer of DNA across the inner membrane may not require the conformational changes necessary for mannose uptake. The stable expression of MceA and MceB offers new

tools to decipher the multiple functions of theE. colimannose transport system.

Acknowledgements

This work was supported by the Roche Research Foun- dation and the State of Geneva. We thank C. Georgopoulos, P. Linder & B. Erni for helpful discussions and plasmids.

Fig. 3. Co-expression of PhoA73-MceA and MceB interferes with mannose metabolism. (A) Strains AB3/pBAD101 (DmanXYZ), DB503/pBAD101 and pBAD33 (man^þ), DB503/pBAD101 and pAB23 (man^þmceB) and DB503/pAB11 and pAB23 (man^þphoA73-mceA mceB) were streaked on minimal M63 medium plates supplemented with 0.2% mannose and the appropriate antibiotics in the absence (left) or in the presence (right) of 0.2% arabinose, and incubated at 37C for 40 h;

all the strains carry theara714deletion. (B) The same strains were streaked on minimal plates supplemented with 0.4% glycerol instead of mannose. (C) The doubling times of DB503/pAB11 and pAB23 (man^þphoA73-mceA mceB) cultures grown in minimal medium supplemented with the indicated sugars were calculated from the slope of optical density increases at 600 nm; the average of duplicate cultures and the range are indicated. (D) Addition of glucose to cells induced in the presence of mannose restores growth.

709 S. Bie´ler et al. / Research in Microbiology 161 (2010) 706e710

(6)

References

Bie´ler, S., Silva, F., Soto, C., Belin, D., 2006. Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

J. Bacteriol. 188, 7049e7061.

Destoumieux-Garzon, D., Thomas, X., Santamaria, M., Goulard, C., Barthelemy, M., Boscher, B., Bessin, Y., Molle, G., Pons, A.M., Letellier, L., Peduzzi, J., Rebuffat, S., 2003. Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization onEscherichia coli. Mol. Microbiol. 49, 1031e1041.

Diep, D.B., Skaugen, M., Salehian, Z., Holo, H., Nes, I.F., 2007. Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proc. Natl. Acad. Sci. U. S. A. 104, 2384e2389.

Elliott, J., Arber, W., 1978. E. coli K-12pelmutants, which block phage lambda DNA injection, coincide withptsM, which determines a component of a sugar transport system. Mol. Gen. Genet. 161, 1e8.

Erni, B., Zanolari, B., Kocher, H.P., 1987. The mannose permease of Escherichia coliconsists of three different proteins. Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA. J. Biol. Chem. 262, 5238e5247.

Esquinas-Rychen, M., Erni, B., 2001. Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol- pyruvate: carbohydrate phosphotransferase system (PTS). J. Mol. Micro- biol. Biotechnol. 3, 361e370.

Guzman, L.M., Belin, D., Carson, M.J., Beckwith, J., 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBADpromoter. J. Bacteriol. 177, 4121e4130.

Lagos, R., Villanueva, J.E., Monasterio, O., 1999. Identification and properties of the genes encoding microcin E492 and its immunity protein. J. Bac- teriol. 181, 212e217.

Lagos, R., Wilkens, M., Vergara, C., Cecchi, X., Monasterio, O., 1993.

Microcin E492 forms ion channels in phospholipid bilayer membrane.

FEBS Lett. 321, 145e148.

de Lorenzo, V., 1984. Isolation and characterization of microcin E492 from Klebsiella pneumoniae. Arch Microbiol. 139, 72e75.

de Lorenzo, V., Martinez, J.L., Asensio, C., 1984. Microcin-mediated interactions betweenKlebsiella pneumoniae andEscherichia colistrains.

J. Gen. Microbiol. 130, 391e400.

de Lorenzo, V., Pugsley, A.P., 1985. Microcin E492, a low-molecular- weight peptide antibiotic which causes depolarization of the Escherichia coli cytoplasmic membrane. Antimicrob. Agents Chemother. 27, 666e669.

Oliver, D.B., Beckwith, J., 1982. Regulation of a membrane component required for protein secretion inEscherichia coli. Cell 30, 311e319.

Thomas, X., Destoumieux-Garzon, D., Peduzzi, J., Afonso, C., Blond, A., Birlirakis, N., Goulard, C., Dubost, L., Thai, R., Tabet, J.C., Rebuffat, S., 2004. Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity. J. Biol. Chem. 279, 28233e28242.

Williams, N., Fox, D.K., Shea, C., Roseman, S., 1986. Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system. Proc. Natl. Acad. Sci. U. S. A. 83, 8934e8938.