APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1994, p. 3809-38140099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Isolation and Characterization of Linocin M18, a BacteriocinProduced by Brevibacterium linens

NATALIA VALDES-STAUBER AND SIEGFRIED SCHERER*Institut fur Mikrobiologie, Forschungszentrum far Milch und Lebensmittel Weihenstephan, Technische Universitat

Munchen, D-85350 Freising, Germany

Received 8 March 1994/Accepted 16 July 1994

Brevibacterium linens M18, isolated from red smear cheese, produces a substance that inhibits the growth ofListeria spp. and several coryneform and other gram-positive bacteria. No gram-negative bacteria were

inhibited. The substance is heat labile, sensitive to proteolytic enzymes, and stable between pH 3 and 12. Highlevels of this bacteriocin, named Linocin M18, were obtained in the stationary growth phase. Linocin M18 was

purified by ultrafiltration, ultracentrifugation, and gel filtration chromatography. In its native form, it is aproteinaceous aggregate with a high molecular weight. Fractions with Linocin M18 activity contained particlesof 20 to 30 nm in diameter. The bacteriocin consists of a single protein subunit with a molecular mass of 31kDa and an isoelectric point of 4.5. N-terminal sequence analysis yielded Met-Asn-Asn-Leu-Tyr-Arg-Glu-Leu-Ala-Pro-Ile-Pro-Gly-Pro-Ala-Ala-Ala-Glu-Ile. Significant homology with published sequences was lacking.

Brevibacteria occur in a number of different habitats, espe-cially in those with high salt concentrations. Apparently, nostrain which can be allocated unequivocally to Brevibacteriumlinens has been isolated from sources other than cheese (19). B.linens contributes to the surface color of Limburger and similarsmear-ripened cheeses and, by its proteolytic activity (13, 14)and production of aroma and flavor constituents, to theripening of such cheeses (18, 22). Red smear cheeses are

manufactured by inoculation of the surface of cheese with a

complex flora of yeasts and coryneform and other bacteriataken from ripened cheese. It is a major disadvantage of thispractice that undesirable spoiling and pathogenic microorgan-isms, e.g., coliform bacteria or Listeria monocytogenes, are

transferred from charge to charge (5, 27, 31). It is, therefore, ofconsiderable interest to develop starter cultures which inhibitundesired microorganisms.

Bacteriocins constitute a quite heterogeneous group ofbacterial exoproteins, protein complexes, or peptides with anantibiotic effect against other than the producer strains. Pres-ently, many attempts are being made to use bacteriocins and/orbacteriocin-producing starters for the preservation of food (8,10, 12). Currently, only Nisin has been granted GenerallyRecognized As Safe status by the Food and Drug Administra-tion. Bacteriocins from a variety of gram-positive species havebeen purified and characterized biochemically as well as

genetically. However, very little is known about bacteriocinsfrom any coryneform bacteria (30, 32).

In a previous study, we isolated a variety of Brevibacteriumand Arthrobacter strains from red smear cheese which inhibitgrowth of L. monocytogenes (32). This report describes thepurification and some properties of the bacteriocin LinocinM18, which is produced by B. linens M18 isolated from thesurface of red smear cheese.

MATERUILS AND METHODS

Bacterial strains and media. The producer strain B. linensM18 and the indicator strain Listeia ivanovii WSLC 3061(Weihenstephan Listeria Collection), which was chosen be-

* Corresponding author. Phone: 49-8161-713516. Fax: 49-8161-714492.

cause of its high-level sensitivity, were grown at 30°C on slantsof plate count agar (Merck, Darmstadt, Germany) and tryp-tose agar (Merck), respectively, and stored at 4°C. For indi-vidual experiments, they were subcultured in the correspond-ing broth media. For sensitivity screening, a variety of bacterialstrains were taken from the Weihenstephan Culture Collectionof the Institute of Microbiology, Forschungszentrum fur Milchund Lebensmittel Weihenstephan (catalog available on re-

quest).Bacteriocin assay. Bacteriocin activity was examined by

spotting 10 ,ul of sterile-filtered samples on agar containing theindicator strain (6 ml of soft tryptose agar was mixed with 0.1ml of a 24-h culture of L. ivanovii WSLC 3061 grown at 30°C,and the mixture, containing approximately 8 x 106 indicatorcells per ml, was poured into a petri dish). Activity was

quantified by serial twofold dilutions (2) and is expressed inactivity units (AU) per milliliter. Sensitivity against a series ofstrains of different groups was tested by the spot on the lawnmethod. Sensitivity tests were repeated at least three times.

Stability of Linocin M18. To evaluate pH stability, a bacte-riocin concentrate (1 ml; 25.6 kAU/ml) was placed on Seph-adex G-25 NAP-10 gel filtration columns (Pharmacia, Uppsala,Sweden) and eluted with Sorensen glycin buffer (pHs 9, 10, 11,and 12), Sorensen potassium phosphate buffer (pHs 6, 7, and8), and Sorensen citrate buffer (pHs 3, 4, and 5) (26). Columnswere preequilibrated with the corresponding buffer. The bac-teriocin was stored in these buffers for 20 h at 4°C. The sampleswere analyzed for the remaining activity after neutralization bythe same procedure but by preequilibrating and eluting theNAP-10 columns with potassium phosphate buffer, pH 7.5. Allexperiments were repeated three times.To determine sensitivity against several enzymatic activities,

concentrated bacteriocin (102.4 kAU/ml after ultrafiltration)was treated at 37°C for 4 h with pronase E, protease VIII,trypsin, proteinase K, ficin, papain, and a-chymotrypsin with a

final concentration of 10 ,ug/ml in 10 mM Tris-HCl at pH 7.5.It was also treated at 37°C for 2 h with lipase, catalase, anda-amylase with a final concentration of 100 ,ug/ml and withRNase and DNase at a final concentration of 10 ,ug/ml inTris-HCl, pH 7.5. Controls consisted of enzymes and ofbacteriocin, respectively. All enzymes were purchased fromSigma, Deisenhofen, Germany.

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Linocin M18 was incubated at different temperatures for upto 30 min under agitation. After incubation, the samples werecooled on ice, and the remaining activity was determined. Abacteriocin concentrate of 6,400 AU/ml was also assayed forstability at room temperature and at 4°C for several days withand without protective substances, such as the protease inhib-itor phenylmethylsulfonyl fluoride (PMSF; 1 mM) and glyc-erol.

Purification of Linocin M18. B. linens M18 was grown to thestationary growth phase in 10 liters of PC broth (Merck) with3% NaCl at 30°C under agitation. Cells were centrifuged(10,000 x gn., for 10 min and at 4°C), the supernatant wascollected and concentrated by ultrafiltration (Sartocon MiniCrossflow System; membrane with a pore size of 20 kDa;Sartorius, Gottingen, Germany) to a final volume of 150 ml.Remaining nucleic acids in the retentate were digested byDNase (8 U/,lJ-Benzon nuclease; Merck) and RNase A (0.5mg; USB, Bad Homburg, Germany) for 6 h at room temper-ature. Of the retentate, 12 ml was placed on top of 15 ml ofsucrose (48% [wt/vol] in 10 mM Tris-HCl, pH 8) in quick-sealcentrifuge tubes (Beckman, Munich, Germany) and ultracen-trifuged for 20 h at 10°C and 257,000 x gm,. The pelletscontaining the bacteriocin activity were resuspended in 3 ml of10 mM Tris-HCl (pH 8) and sterile filtered. Final purificationwas performed by fast protein liquid chromatography gelfiltration chromatography (Superose 6 column; Pharmacia) in10 mM Tris-HCl, pH 8. Active fractions were concentrated bycentrifugation under vacuum. Protein concentrations weredetermined by microassay (Bio-Rad Laboratories GmbH, Mu-nich, Germany) with bovine serum albumin (BSA) as a stan-dard.

Electrophoresis. Discontinuous sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) (21) wasperformed at 10°C in Tris-glycine at 25 mA in a MidgetElectrophoresis Unit (LKB 2050; Pharmacia) by following theinstructions provided by the manufacturer. Proteins were fixedwith 40% methanol-10% acetic acid and silver stained by themethod of Blum et al. (4). For isoelectric focusing, immobi-lized pH gradient gels in a horizontal system supplementedwith small amounts of carrier ampholytes (IPG-CA gels) wereused (24). For two-dimensional electrophoresis, individual gelstrips were cut from the first-dimension IPG-CA gels, directlyequilibrated for SDS-PAGE, and then transferred onto thesurface of a horizontal, discontinuous ultrathin-layer SDS gel(ExcelGel; 8 to 18% total acrylamide concentration; Pharma-cia).Western blotting (immunoblotting), amino acid composi-

tion, and amino-terminal sequence. Proteins were separatedon discontinuous SDS-polyacrylamide gels and then trans-ferred to polyvinylidene difluoride membranes (Immobilon P;Millipore, Eschborn, Germany) by semidry, discontinuoushorizontal electroblotting at 0.8 mA/cm2 for 60 min. Mem-branes were stained with Coomassie blue R-350 (Pharmacia)for 10 min destained in 90% methanol-7% acetic acid for 30 s,air dried, and stored at -70°C. Bands of interest were excisedand used for Edman degradation in a protein sequencer(Applied Biosystems model 477 A). For amino acid composi-tion analysis, bands were destained completely in 95% meth-anol. Proteins were hydrolyzed in 6 N HCl at 110°C for 24 hand dried under vacuum. Amino acids were converted to theirDABSYL derivatives and analyzed by reversed-phase high-pressure liquid chromatography HPLC (23).

Electron microscopy. Electron microscopy was performedwith purified bacteriocin fractions after gel filtration on Super-ose 6. Bacteriocin suspensions were adsorbed to carbon-coatedFormvar films on copper grids, stained with uranyl acetate (2%

[wt/vol], pH 4.2; Merck), and investigated with a Zeiss EM-1OA transmission electron microscope (34).

RESULTS

Inhibitory spectrum. The inhibitory activity was not re-stricted to closely related strains, but gram-negative bacteriawere insensitive. Sensitive and insensitive strains are shown inTable 1. Generally, single species comprised sensitive as well asinsensitive strains. The producer strain was immune. Sensitivityof Listeria spp. was generally very high; 86.8% of 91 testedstrains were inhibited. In particular, 90.5% of 42 tested L.monocytogenes strains, 91.2% of 34 L. innocua, and 63.6% of11 L. ivanovii were inhibited by B. linens M18 (32).

Stability of Linocin M18. Linocin M18 is sensitive against anumber of proteases, which reveals its proteinaceous nature.Activity after incubation in the presence of proteases de-creased from 102 kAU/ml to 0.2 to 3.2 kAU/ml. No inhibitionof bacteriocin activity by nucleases, lipase, catalase, o-amylase,and the proteases ficin and papain was observed.

Linocin M18 was subjected to a 24-h treatment at 4°C at pHvalues from 3 to 12. Activity was completely conserved be-tween pH 5 and 9 and diminished only slightly at pH 3 to 4 and10 to 12. Chloroform (10%), ethanol (20 and 30%), andacetone (20 and 30%) were added to bacteriocin samples for 1h and then removed by evaporation under vacuum, and theremaining activity was quantified. Ethanol, acetone, or chloro-form had no effect (data not shown).

Linocin M18 was heat sensitive. After 5 min at 80°C, noactivity was detectable even in a highly concentrated sample(102.4 kAU/ml). At temperatures between 40 and 50°C, it wasstable up to 30 min. By freeze drying and storage at -20°C for1 year and at 4°C for at least 4 days, no decrease of activity wasfound in the partially purified bacteriocin sample after ultra-filtration. At room temperature, the activity diminished rapidlyafter 1 to 2 days, and no difference was observed when aprotease inhibitor (PMSF) or glycerol was added.

Purification of Linocin M18. Density characteristics of Li-nocin M18 were used in the purification procedure. Aftercentrifugation of the bacteriocin sample on a preformedsucrose gradient (26 to 46% [wt/vol]), most of the activity wasfound at a sucrose concentration of between 36 and 38% (Fig.1); this concentration corresponds to a sucrose density ofapproximately 1.16 g/cm3. Accordingly, when Linocin M18 wasultracentrifuged in a 28% (wt/vol) sucrose solution, activity wasfound in the pellet. This pellet was separated by gel filtration(Fig. 2). The bacteriocin always eluted near the void volume.This purification procedure yielded low recovery but highspecific activity and purity (Table 2).

Estimation of the native molecular weight by gel filtrationwas not possible because the activity eluted in the void volumeof the Superose 6 column. This was also found when theoriginal culture supernatant, which was not treated previously,was separated on Superose 6. When the gel filtration fractionscontaining the bacteriocin were observed under the electronmicroscope, particles of a size between 20 and 30 nm werefound (Fig. 3). To check whether disaggregation of LinocinM18 was possible, the bacteriocin was eluted from the gelfiltration column with buffer to which different agents such as0.5% Tween 20, 2 M NaCl, 50 mM dithiothreitol, or 6 M ureawas added. Fractions were tested for activity. Bacteriocinsamples were also treated with EDTA at a final concentrationof 8 mM, and before bioassay was performed, CaCl2 was addedto neutralize the excess EDTA. Controls consisted of thebacteriocin samples in the dilutions used and of EDTA andCaCl2 without bacteriocin. No disaggregation was achieved by

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TABLE 1. Sensitivity of various bacteria against Linocin M18

No. of strains i No. of strainsSpecies iI Species

Sensitive Insensitive .Sensitive Insensitive

Bacillus lentusBacillus polymyxaBacillus lentusBacillus circulansBacillus cereusBacillus alveiBacillus subtilisBacillus pumilisBacillus liqueniformisBacillus laterosporusBacillus firmusBacillus coagulansBacillus brevisArthrobacter sp.Arthrobacter nicotianaeArthrobacter parafineusArthrobacter picolinophilusArthrobacter hydrocarboglutamicusArthrobacter rubellusArthrobacter albidusArthrobacter oxamicetus subsp. propioniphenicolusArthrobacter atrocyaneusArthrobacter aurescensArthrobacter citreuArthrobacter crystallopoietesArthrobacter globiformisArthrobacter histidinolovoransArthrobacter ilicisArthrobacter luteusArthrobacter oxydansArthrobacter pascensArthrobacter polychromogenesArthrobacter protophormiaeArthrobacter ramosusArthrobacter simplexArthrobacter stabilisArthrobacter sulfureusArthrobacter uratoxidansArthrobacter variabilisAureobacterium flavescensAcetobacter pasteurianusAcetobacter rancensAcinetobacter calcoaceticusAcinetobacter calcoaceticusActinimadura maduraeAeromonas hydrophila subsp. anaerogenesAlcaligenes toleransBrevibacterium linensBrevibacterium lactofermentumBrevibacterium caseiBrevibacterium flavumBrevibacterium manisBrevibacterium helvolumBrevibacterium albumBrevibacterium chang-fua

1 1I

131222111152

11214111211211121111122121

131

1114

151

111

111

2112

1

Brevibacterium oxydansBrevibacterium ammoniagenesBrevibacterium divaricatumBrevibacterium fermentansBrevibacterium imperialeBrevibacterium lyticumBrevibacterium saccharolyticumCitrobacter freundiiCorynebacterium sp.Corynebacterium fasciansCorynebacterium ammoniagenesCorynebacterium glutamicumCorynebacterium flavescensCorynebacterium insidiosumCorynebacterium liquefaciensCorynebacterium mycetoidesCorynebacterium variabilisCurtobacterium luteumEnterobacter aerogenesEnterobacter cloacaeEnterococcus faecalisEscherichia coliErwinia nignfluensGluconobacter oxydansKlebsiella aerogenesKluyvera citrophilaLactobacillus brevisLactobacillus acidophilusLactobacillus caseiLactobacillus delbrueckii subsp. bulgaricusLactobacillus helveticusLactobacillus plantarumLactobacillus lactisLactococcus lactisLeuconostoc mesenteroidesMicrobacterium lacticumMicrococcus luteusMicrococcus roseusMycobacterium violaceumPediococcus sp.Propionibacterium freudenreichiiPropionibacterium acidipropiociciProteus mirabilisProteus mirabilisPseudomonas aeruginosaPseudomonas alcaligenesPseudomonas diminutaPseudomonas fluorescensPseudomonas saccharophilaRhodococcus sp.Rhodococcus fasciansStaphylococcus aureusStaphylococcus caseolyticusStreptococcus salivariusVibrio proteolyticus

I 15

21

43 31 3

2 1

31

31

2

14

24

11

1

1

1

2

31

1

these treatments, since Linocin M18 eluted always near thevoid volume.

After gel filtration, the bacteriocin appeared as a single bandwith a molecular mass of approximately 31 kDa in silver-stained SDS-polyacrylamide gels (Fig. 4). A two-dimensionalelectrophoresis of purified bacteriocin showed one spot withan isoelectric point of 4.5 (data not shown). The amino acidcomposition of Linocin M18 is shown in Table 3. After Linocin

M18 was blotted to a polyvinylidene difluoride membrane andafter Edman degradation, the following N-terminal amino acidsequence was found: Met-Asn-Asn-Leu-Tyr-Arg-Glu-Leu-Ala-Pro-Ile-Pro-Gly-Pro-Ala-Ala-Ala-Glu-Ile. Linocin M18contains a methionine residue at the N terminus, and the Nterminus is rich in alanine (4 of 19) and in proline (3 of 19). Nosignificant sequence similarity to any protein recorded in theProtein Identification Resource Database (release 37.0, June

i-

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TABLE 2. Purification steps of Linocin M18'

Vol Protein Activity Sp act YieldFraction (ml) (mg/ml) (AU/ml) (AU/mg) (%)

Retentateb 150 6.8 12,800 1,882 100Pellet' 3.0 2.8 12,800 4,571 2Gel filtration 1.5 0.2 3,200 16,000 0.25

a cf. Fig. 4.b From ultrafiltration.c From ultracentrifugation.

28 30 32 34 36 38 40 42Sucrose concentration (%)

FIG. 1. Distribution of Linocin M18 in a preformed sucrose gradi-ent (26 to 46% [wt/vol] in Tris [10 mM; pH 8]). A 1-ml sample (6,400AU/ml) was centrifuged for 64 h and at 10°C at 130,000 x gm: (rotorSW 28.1; Beckman). Total percent recovery of activity was approxi-mately 44%.

1993; compiled by National Biomedical Research Foundationand distributed by Hitachi Software Engineering America,Ltd.) was found.

DISCUSSIONIn this paper, the isolation and purification of the new

bacteriocin Linocin M18 produced by B. linens M18 aredescribed. A variety of inhibitory substances, however, can beproduced by bacteria. In this case, it can be excluded thatorganic acids or hydrogen peroxide causes the inhibitionobserved: B. linens does not produce acids from carbohydrateswhen growing in peptone medium (19), the pH in the growthmedium was in the range of 7.5 to 8.5, B. linens is catalasepositive, and the molecular weight of the inhibitory activity wasvery large, as demonstrated by ultrafiltration and gel filtration.Protease sensitivity is a key criterion for the characterization ofan inhibitory substance such as a bacteriocin. Linocin M18 is

01,04

_ -

0(1,0300

O 0,02D0 A

10 15Elutioii volume (ml)

sensitive to several proteases, including trypsin, demonstratingits proteinaceous nature; participation of other substancessuch as phosphorus, lipids, or sugars in the inhibitory complex,as was shown, for example, for Staphylococcin 414 (11),Staphylococcin 1580 (17), or Pediocin SJ-1 (29), cannot beexcluded. According to the recent classification of Klaenham-mer (20) for bacteriocins produced by lactic acid bacteria,Linocin M18 would belong to either class III or IV.

Native Linocin M18 forms aggregates of extremely highmolecular masses (>2,000 kDa). Other bacteriocins of thatsize range have been described: Staphylococcin 414 (11),Megacin Cx (9), and Lactacin F (25). An association withmembrane vesicles has been suggested in these cases, andBecker et al. (3) provided evidence for the association of thebacteriocin activity of Thermus rubens with membrane vesicles.The density calculated for Linocin M18 is comparable to thatof membrane vesicles and protein aggregates. Its isoelectricpoint of 4.5 as well as the molecular mass of 31 kDa of thesubunit is not unusual compared with those of other bacterio-cins produced by gram-positive bacteria (1, 16, 30). A struc-tural similarity to defect bacteriophages, on the other hand,was not found for Linocin M18.The existence of a large number of diverse microbial antag-

onistic substances such as bacteriocins is now well recognized(7, 20, 28, 33). Usually, bacteriocins inhibit only closely relatedbacteria. One exception is Nisin, which inhibits strains ofvarious gram-positive genera, including the spore formers

20 25

FIG. 2. Elution profile of Linocin M18 from the gel filtration column Superose 6 after ultracentrifugation. Shaded fractions contain LinocinM18 activity. Results of SDS-PAGE of the corresponding fractions are shown in the inset.

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TABLE 3. Amino acid composition of Linocin M18

Amino acid mol%"

Asp.5.3Glu.10.1Ser.7.7Thr.6.2Gly.11.0Ala.13.5Arg.4.3Pro.5.1Val.9.0Met.NDIle.7.4Leu.12.1Phe.3.3Lys.1.4His.1.7Tyr.1.9Cys.ND

FIG. 3. Transmission electron micrograph of globular structuresobserved in the gel filtration fraction with Linocin M18 activity (Bar,100 nm).

Bacillus and Clostridium. Similar to Nisin, Linocin M18 exhib-its an extraordinarily wide activity spectrum which includesstrains of more than one genus. The interaction with thegrowth of Listeria spp. (31, 32), however, is most important forapplied purposes. Biopreservation systems using bacteriocinshave gained increasing attention as a means of natural controlof the growth of pathogenic and spoilage organisms in food-stuff (6, 15, 16). However, except for the Linocin M18 of thisstudy, no bacteriocins have been isolated from any coryneform

M A B C

kDa

66

45

36

29

24

20,1

14,2

_ 009

FIG. 4. Purification steps and estimation of the molecular mass ofthe Linocin M18 monomer. Lane A, retentate after ultrafiltration; laneB, pellet-containing bacteriocin activity; lane C, active fraction aftergel filtration chromatography. lane M, molecular mass standards(bovine lactalbumin, 14.2 kDa; trypsin inhibitor, 20.1 kDa; trypsino-gen, 24 kDa; carbonic anhydrase, 29 kDa; GAL-3-phospho dehydro-genase, 36 kDa; ovalbumin, 45 kDa; and BSA, 66 kDa).

a ND, not detected. Although the N-terminal amino acid sequence contains aMet residue, its moles percent in the 31-kDa protein may fall under the detectionlimit.

bacteria. This paper is only a first step towards characterizingbacteriocins of coryneform bacteria and exploring their poten-tial use in the biological control of pathogenic Listeria spp. inspecialized dairy products such as red smear cheese.

ACKNOWLEDGMENTS

N.V.-S. thanks FICYT (Asturias, Spain) for financial support until1993.Thanks are due to M. J. Loessner for valuable discussions and

helpful support in the laboratory. Amino acid analysis was done by I. B.Krause at the Institute of Chemistry, FML (Freising, Germany).Amino acid sequencing was done by the protein sequencing facility ofVirginia Tech., Blacksburg. H. C. Bartscherer (Freising, Germany)provided the facility for electron microscopy, R. Zink supportedgreatly in electron microscopical work, and A. Weise was of great helpin preparing the manuscript.

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