Instituto de Productos Lácteos de Asturias (IPLA-CSIC), 33300-Villaviciosa, Asturias, Spain1
Departamento de Biología Funcional, Area de Microbiología, Universidad de Oviedo, 33006-Oviedo, Asturias, Spain2
Author for correspondence: Juan E. Suárez. Tel: +34 98 510 35 59. Fax: +34 98 510 31 48. e-mail: jsuarez{at}sauron.quimica.uniovi.es
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ABSTRACT |
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Keywords: lactococcin 972, septum biosynthesis, bacteriocin, Lactococcus lactis
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INTRODUCTION |
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The potential application of bacteriocins of lactic acid bacteria as food preservatives requires an in-depth knowledge of how they exert their bactericidal effect. Most bacteriocins whose primary mode of action is known act at the plasma membrane. It has been proposed that these peptides form poration complexes that traverse the phospholipid bilayer. This provokes membrane permeabilization and hence depletion of the proton-motive force of sensitive cells (Ojcius & Young, 1991 ; Driessen et al., 1995
; Abee, 1995
).
In some cases, the pore only allows leakage of ions, mainly K+ and, in the cases in which this has been tested, inorganic phosphate (Parente et al., 1996 ; McAuliffe et al., 1998
; Barrena-González et al., 1996
; Moll et al., 1998
). The depletion of phosphate presumably causes a shift in the ADP/ATP intracellular equilibrium leading to ATP hydrolysis and cell de-energization (Abee et al., 1994
). In other cases, the size of the pores may increase, thus allowing the subsequent release of larger molecules such as amino acids and even ATP (González et al., 1996
; Zajdel et al., 1985
; Maisnier-Patin et al., 1996
).
In contrast, the type B lantibiotics mersacidin and actagardine, which are globular peptides with no amphipathic properties, kill susceptible cells by interfering with cell wall biosynthesis, namely at the level of transglycosylation, the reaction that mediates integration of new monomers into pre-existing peptidoglycan (Brötz et al., 1998 ).
The plasma membrane was found not to be the primary target of lactococcin 972. Sensitive cells treated with the bacteriocin did not suffer either leakage of cytoplasmic solutes or significant inhibition of macromolecular synthesis, which would be immediately observed upon loss of the proton-motive force (Martínez et al., 1996 ; González et al., 1996
). Furthermore, lactococcin 972 is not a hydrophobic peptide, a typical feature of pore-forming bacteriocins, as was deduced from its amino acid sequence (Martínez et al., 1999
). This bacteriocin is also unusual because its functional form is a homodimer whose components are weakly bound; they can be separated by heating to 50 °C or by acidic pH, the resulting monomers being devoid of any antibacterial activity (Martínez et al., 1996
).
In this report, we present data indicating that the primary target of lactococcin 972 is the inhibition of septum formation in susceptible lactococci. This results in deformation of the cells as visualized by optical microscopy, and subsequently in gross structural changes that lead to cell death.
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METHODS |
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Bacteriocin purification and quantification.
Lactococcin 972 was extracted from supernatants of late-exponential-phase cultures of L. lactis IPLA 972 as previously described (Martínez et al., 1996 ). The protein concentration was determined with the Bio-Rad Protein Assay kit. The bacteriocin activity was quantified by the agar diffusion test (Reddish, 1929
). Twofold serial dilutions were tested and the highest one that produced a clear zone of growth inhibition on the indicator lawn was defined as 1 arbitrary unit of activity ml-1 (AU ml-1), which corresponds to a lactococcin 972 concentration of 12·7 nM.
Cell wall biosynthesis measurements.
Synthesis of peptidoglycan in exponentially growing cultures of L. lactis MG1614 (OD600 0·2) was followed by measuring the continuous incorporation of N-acetyl-D-[1-3H]glucosamine (192 GBq mmol-1, 37 MBq ml-1), at a final concentration of 37 kBq ml-1, into trichloracetic-acid-insoluble material. Half of the cultures were treated with purified lactococcin 972 (50 AU ml-1), which was replaced in the controls with the same volume of 50 mM sodium phosphate buffer (pH 7·0). Incubation was at 32 °C and proceeded for 180 min. Samples were collected at intervals and processed as described previously (Rodríguez et al., 1986 ).
Optical and electron microscopy.
Samples were taken from exponentially growing cultures of L. lactis MG1614 before and after (up to 120 min) addition of lactococcin 972 (50 AU ml-1). Light micrographs were taken with an Olympus BH-2 phase-contrast microscope. For negatively stained and thin-section electron microscopy, cells were harvested by centrifugation and washed twice in sterile distilled water. Cell suspensions were negatively stained with aqueous 1% uranyl acetate (pH 4·0). For thin-section preparation, washed cells were embedded in 2% Noble agar (Difco), fixed overnight in 1% osmium tetraoxide in sodium diethylbarbiturate/acetate buffer (pH 6·0) (Ryter & Kellenberger, 1958 ), and stained with 2% uranyl acetate in the same buffer for 2 h. Dehydration was done in a graded acetone series (25100%), and embedding in Epon 812. Sections were stained with 2% uranyl acetate in ethanol and Reynolds lead citrate (Reynolds, 1963
). Negatively stained samples and thin sections were observed with a JEOL 2000 EX-II electron microscope operating at 80 kV.
Flow cytometry analysis.
Early-exponential-phase cultures (OD600 0·2) of L. lactis MG1614 were divided into aliquots of 5 ml. Purified lactococcin 972 (50 AU ml-1) or the same volume of 50 mM sodium phosphate buffer (pH 7·0) were added to the test and control samples, respectively. Culture aliquots (0·5 ml) were collected at intervals, centrifuged, washed twice with phosphate-buffered saline (pH 7·0) (PBS) and resuspended in 0·5 ml PBS containing 5 µg ml-1 propidium iodide (Sigma). Samples were allowed to stain for 5 min at room temperature before being analysed by flow cytometry. The stock solution was prepared by dissolving the propidium iodide to a final concentration of 1 mg ml-1 in ethanol, immediately prior to use. The flow cytometer used was a Bryte-HS (Bio-Rad) equipped with WinBryte software (Bio-Rad). Ilumination was provided by a 75 W lamp.
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RESULTS |
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DISCUSSION |
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Septum formation is a complex process, best studied in Escherichia coli, in which the products of at least nine genes are involved (Lutkenhaus & Addinall, 1997 ). The process occurs in two stages. In the initial stage, a distant homologue of eukaryotic tubulin, FtsZ, forms the so-called cytokinetic ring, which is believed to be responsible for the constriction that the cell suffers at the septum region (Erickson et al., 1996
; Mukherjee & Lutkenhaus, 1994
). In the second stage, which is sensitive to ß-lactam antibiotics, the transpeptidase PBP3, which is the product of gene ftsI, mediates formation of the cross-links between the peptidoglycan strands at the growing septum (Wang et al., 1998
; Botta & Park, 1981
). Cells devoid of FtsZ or PBP3 form smooth and blunt constricted multinucleate filaments, respectively, indicating that FtsZ is needed to initiate constriction while PBP3 is important for septum invagination (Pogliano et al., 1997
). If L. lactis has a septum formation machinery similar to that of E. coli, we must conclude that lactococcin 972 does not affect the initial stage of the process, since cells treated with the bacteriocin show equatorial constriction and even septum primordia. It presumably rather inhibits one or more steps at the second stage, i.e. septum invagination. We might speculate that lactococcin 972 affects PBP3 function. However, this protein, at least in E. coli, interacts with many others, such as FtsA, FtsQ and FtsW (Wang et al., 1998
; Tormo et al., 1986
). Thus, any of these might be the target of the bacteriocin. In fact, some differences are observed with respect to E. coli ftsI mutants. For example, treated L. lactis cells do not form long filaments, although they suffer an elongation that precedes macromolecular synthesis arrest and death of the cell.
This might be due to the different patterns of cell wall growth shown by rod-shaped bacteria and cocci. In the first case, exemplified by E. coli and Bacillus subtilis, longitudinal growth is effected by deposition of new wall monomers all along the rod. In contrast, septum formation is a localized process in which an annular disk of wall material is produced inwards to bisect the two daughter cells (de Chastelier et al., 1975 ; Archibald et al., 1993
). Therefore, inhibition of septum formation may not necessarily impair elongation of the cells, which would result in the formation of filaments. In cocci, both elongation and septum formation take place at an annular band located in the equatorial zone of the cell (Cole & Hahn, 1962
). This band splits during growth and the resulting bands move away as new material is placed between them. The septum is initiated at this single starting point and proceeds via centripetal growth (Daneo-Moore & Shockman, 1977
; Archibald et al., 1993
). In other words, cell elongation and septum formation are physically and, possibly, functionally linked in Gram-positive cocci. It is thus conceivable that inhibition of septum formation would affect cell growth as well. This might trigger deformation and lysis of the cells, especially if the autolysins that promote wall turnover and cell separation (Wong et al., 1974
; Koch & Doyle, 1986
) are not inhibited.
Most of the data on lactococcin 972 indicate that it is an unusual bacteriocin. In contrast to virtually all antimicrobial peptides from Gram-positive bacteria, its active form is a homodimer, and it is sensitive to heat and low pH (Martínez et al., 1996 ). Furthermore, it is probably exported via a Sec-dependent mechanism (Martínez et al., 1999
) and acts on cell wall biosynthesis, not, as do most bacteriocins, by opening pores in the plasma membrane. Recently, a new bacteriocin, enterolysin A, which is homologous to different cell-wall-lytic proteins, has been described (Nilsen et al., 1999
). Although its mode of action has not yet been studied, the cell wall might be its target. It might hence be reasonable to consider the creation of a new subgroup of bacteriocins among those already described in class II of Nes et al. (1996)
, to include bacterocins whose target is the cell wall. Further studies on the primary target of lactococcin 972 are in progress in order to assign it definitely to the subgroup suggested above.
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ACKNOWLEDGEMENTS |
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This work was financially supported by grants ALI93-0873-C02 and ALI97-0658-C03 from the Comisión Interministerial de Ciencia y Tecnología of Spain. B.M. was the recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia of Spain.
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Received 29 July 1999;
revised 24 December 1999;
accepted 10 January 2000.