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2,5-Diketopiperazines Produced by Bacillus pumilus During Bacteriolysis of Arthrobacter citreus

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ORIGINAL ARTICLE 2,5-Diketopiperazines Produced by Bacillus pumilus During Bacteriolysis of Arthrobacter citreus Christiane Brack & Annett Mikolasch & Frieder Schauer Received: 23 May 2013 /Accepted: 10 October 2013 # Springer Science+Business Media New York 2014 Abstract We report the detection by gas chromatography/ mass spectrometry and liquid chromatography/mass spec- trometry analyses of the secreted 2,5-diketopiperazines (DKPs) cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro), cyclo(-Ile-Pro), cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(- HyP-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro) produced by Bacillus pumilus . The study focuses on a marine isolate and a laboratory test strain of B. pumilus with capabilities to lyse pregrown living cell lawns of different bacterial species, among them Arthrobacter citreus . Chromatographic methods were used to analyze induced bioactive compounds. At least 13 different DKPs are produced by B. pumilus . Both strains respond with an increased production of the DKPs cyclo(- Gly-Pro), cyclo(-Ala-Pro), and cyclo(-Val-Pro) to the presence of pasteurized A. citreus cells after 4 h in a nutrient-poor liquid medium. In agar diffusion assays, these DKPs did not cause lysis zones in living cell lawns, but they did inhibit further growth of several pregrown test bacteria in microplates even at concentrations as low as 1 μg ml -1 . Antibiotic substances produced by B. pumilus after 20 h of cultivation in a special lysis medium showed lytic activity in cell-free extracts of B. pumilus culture supernatants. Keywords Cyclic peptides . Bacterivory . Intraguild predation . Lysobacter Introduction The degradation of bacterial cells, also called bacteriolysis, is a fundamental process during saprobiosis (degradation of dead cells) and predation (bacterivory, ingestion of living bacteria as an energy supply) within natural bacterial commu- nities. In contrast to saprobiosis, predation needs an initial attack before cell matter can be disposed of by the predator. Predation is very common, and during its long evolutionary history, different predation strategies have been developed by diverse predatory bacterial species (Martin 2002; Jurkevitch and Davidov 2007). Recently, the definition of bacterial pre- dation has been extended to so-called intraguild predators like Streptomyces spp. and Escherichia coli , which produce anti- biotics or bacteriocins with which they may predate on direct food competitors (Berleman and Kirby 2009; Leisner and Haaber 2012). Predation is a more complex process than saprobiosis, but bacteriolysis always depends on extracellular enzymes and also usually on bioactive metabolites which cause decompo- sition both of the bacterial cell surface and of its cell contents. Bacteriolytic substances which permeabilize or destroy cell surface structures might be useful as biosurfactants, or as antimicrobial control agents, for example, in aquaculture and in agriculture, as disinfectants for surface treatment, or for recovering intracellular products from yeasts or bacteria (Fiolka and Witkowski 2004; Folman et al. 2004). For this reason, research on bacteriolysis can provide access to new antibacterial agents and bacteriolytic enzymes (enzybiotics), biosurfactants, and new antibiotics (Parisien et al. 2008; Xie et al. 2012). In the past, research on antagonistic marine bacteria fo- cused on applicable secondary metabolites following stan- dard screening procedures, and interspecific interactions have rarely been studied in an ecological context (Grossart et al. 2004 ). However, the production of bioactive Electronic supplementary material The online version of this article (doi:10.1007/s10126-014-9559-y) contains supplementary material, which is available to authorized users. C. Brack (*) : A. Mikolasch : F. Schauer Department of Applied Microbiology, Institute of Microbiology, University Greifswald, Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald, Germany e-mail: [email protected] Mar Biotechnol DOI 10.1007/s10126-014-9559-y
Transcript

ORIGINAL ARTICLE

2,5-Diketopiperazines Produced by Bacillus pumilusDuring Bacteriolysis of Arthrobacter citreus

Christiane Brack & Annett Mikolasch & Frieder Schauer

Received: 23 May 2013 /Accepted: 10 October 2013# Springer Science+Business Media New York 2014

Abstract We report the detection by gas chromatography/mass spectrometry and liquid chromatography/mass spec-trometry analyses of the secreted 2,5-diketopiperazines(DKPs) cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro),cyclo(-Ile-Pro), cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(-HyP-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro) producedby Bacillus pumilus. The study focuses on a marine isolateand a laboratory test strain of B. pumilus with capabilities tolyse pregrown living cell lawns of different bacterial species,among them Arthrobacter citreus. Chromatographic methodswere used to analyze induced bioactive compounds. At least13 different DKPs are produced by B. pumilus. Both strainsrespond with an increased production of the DKPs cyclo(-Gly-Pro), cyclo(-Ala-Pro), and cyclo(-Val-Pro) to the presenceof pasteurized A. citreus cells after 4 h in a nutrient-poor liquidmedium. In agar diffusion assays, these DKPs did not causelysis zones in living cell lawns, but they did inhibit furthergrowth of several pregrown test bacteria inmicroplates even atconcentrations as low as 1 μg ml−1. Antibiotic substancesproduced by B. pumilus after 20 h of cultivation in a speciallysis medium showed lytic activity in cell-free extracts ofB. pumilus culture supernatants.

Keywords Cyclic peptides . Bacterivory . Intraguildpredation . Lysobacter

Introduction

The degradation of bacterial cells, also called bacteriolysis, isa fundamental process during saprobiosis (degradation ofdead cells) and predation (bacterivory, ingestion of livingbacteria as an energy supply) within natural bacterial commu-nities. In contrast to saprobiosis, predation needs an initialattack before cell matter can be disposed of by the predator.Predation is very common, and during its long evolutionaryhistory, different predation strategies have been developed bydiverse predatory bacterial species (Martin 2002; Jurkevitchand Davidov 2007). Recently, the definition of bacterial pre-dation has been extended to so-called intraguild predators likeStreptomyces spp. and Escherichia coli, which produce anti-biotics or bacteriocins with which they may predate on directfood competitors (Berleman and Kirby 2009; Leisner andHaaber 2012).

Predation is a more complex process than saprobiosis, butbacteriolysis always depends on extracellular enzymes andalso usually on bioactive metabolites which cause decompo-sition both of the bacterial cell surface and of its cell contents.Bacteriolytic substances which permeabilize or destroy cellsurface structures might be useful as biosurfactants, or asantimicrobial control agents, for example, in aquaculture andin agriculture, as disinfectants for surface treatment, or forrecovering intracellular products from yeasts or bacteria(Fiolka and Witkowski 2004; Folman et al. 2004). For thisreason, research on bacteriolysis can provide access to newantibacterial agents and bacteriolytic enzymes (enzybiotics),biosurfactants, and new antibiotics (Parisien et al. 2008; Xieet al. 2012).

In the past, research on antagonistic marine bacteria fo-cused on applicable secondary metabolites following stan-dard screening procedures, and interspecific interactionshave rarely been studied in an ecological context (Grossartet al. 2004). However, the production of bioactive

Electronic supplementary material The online version of this article(doi:10.1007/s10126-014-9559-y) contains supplementary material,which is available to authorized users.

C. Brack (*) :A. Mikolasch : F. SchauerDepartment of Applied Microbiology, Institute of Microbiology,University Greifswald, Friedrich-Ludwig-Jahn-Str. 15,17487 Greifswald, Germanye-mail: [email protected]

Mar BiotechnolDOI 10.1007/s10126-014-9559-y

metabolites sometimes requires induction by small amountsof other bacterial cells or their metabolites and is weak orundetectable in the pure cultures normally used (Burgesset al. 1999).

The present study focuses on the secretion of bioactivemetabolites induced in two strains of Bacillus pumilus bycells of Arthrobacter citreus present in the culture medi-um. Former studies on B. pumilus strains documentedtheir competency to lyse different living bacterial specieson agar plates and their lysis activity against pasteurizedand living A. citreus cells in liquid culture (Brack et al.2013). In order to understand the mechanisms by whichB. pumilus degrades both living and dead cells, an isolateof B. pumilus from the Baltic Sea (SBUG 1800) and alaboratory strain (SBUG 1921) have been cultivated inliquid medium in the presence of pasteurized cells ofA. citreus. Agar diffusion assays of cell-free supernatantextracts from these cultures were carried out to decipherthe bacteriolytic mechanism, and the supernatants wereanalyzed chromatographically to separate potentially bac-ter iolyt ic metabol i tes for ident i f icat ion by gaschromatography/mass spectrometry (GC/MS) and liquidchromatography/mass spectrometry (LC/MS). The inhibi-tory activity of the potentially bacteriolytic metaboliteswas analyzed against different Gram-positive and Gram-negative bacteria to determine their applicability as anti-microbial agents.

Material and Methods

Organisms

The marine isolate B. pumilusSBUG 1800 formally describedas 7 A (Brack et al. 2013) was isolated from the surface ofestuarine brackish waters of the Southern Baltic Sea at the siteof the Rassower Strom. B. pumilus SBUG 1921 (Brack et al.2013) was obtained from the strain collection of the Depart-ment of Microbial Physiology and Molecular Biology of theInstitute of Microbiology in Greifswald.

Antibacterial activity of the diketopiperazines was testedagainst 12 strains of bacteria: six Gram-positives (A. citreusSBUG 321, B. pumilus SBUG 1800, Bacillus subtilis subsp.spizizenii SBUG 14, B. subtilis subsp. subtilis DSM 10 T/SBUG 226, Micrococcus luteus SBUG 16, Staphylococcusaureus SBUG 1134) and six Gram-negatives (Aeromonashydrophila SBUG 1762, E. coli SBUG 13, Pseudomonasaeruginosa SBUG 6, Pseudomonas putida SBUG 24, Vibriocholerae SBUG 1612, Vibrio ordalii SBUG 1865/DSM19621). Among the 12 tested bacteria, there are four faculta-tive human pathogens (E. coli, P. aeruginosa, S. aureus,V. cholerae) and two fish pathogenic species (A. hydrophilaand V. ordalii).

Culture Media and Methods

Nutrient broth II (SIFIN, Berlin) and Bacto Marine Broth(DifcoTM, Otto Nordwald, Hamburg) served as cultivationmedia. Lysis medium (LM) containing pasteurized A. citreuscells was used to induce antimicrobial behavior in B. pumilus.It was prepared by growing A. citreuscells in nutrient broth for24 h at 30 °C with constant shaking at 180 rpm. One hundredmilliliters of this preculture was used to inoculate 1,000 ml ofsterile nutrient broth in a 3,000-ml Erlenmeyer flask. After48 h of growth, the cells were centrifuged (20 min, 4 °C,15,000×g) and the pellets were pasteurized for 30 min at65 °C. The liquid LM consists of a mineral salt medium forbacteria (Schauer 1981) supplemented with 0.125 % biotinstock solution (0.0164 mM) and 10 g l−1 of the pasteurizedA. citreus cell pellet as sole substrate. The same mediumcontaining 0.1 % glucose instead of bacterial cells served asa lysis control medium (LCM). One milliliter of an overnightculture of B. pumiluswas inoculated into 50 ml LM or LCMand incubated in 500 ml shaking flasks at 30 °C. After 4 and20 h, samples of these cultures were centrifuged for 30 min ina refrigerated superspeed centrifuge (Sorvall® RC-5B,Dupont Instruments) at 4 °C and 15,000×g. Thus, for eachB. pumilus strain, four different culture supernatant sampleswere obtained: LM (4 h), LCM (4 h), LM (20 h), and LCM(20 h). At least two independent culture experiments werecarried out for B. pumilusSBUG 1800 and four for B. pumilusSBUG 1921. As controls, the two media (LM and LCM)without an inoculation with B.pumiluswere prepared the sameway. To prepare dry extracts for agar diffusion assays, 40ml ofeach sample was lyophilized.

Gas Chromatography/Mass Spectrometry and LiquidChromatography/Mass Spectrometry Analyses

Solid-Phase Extraction

For both of these chromatography methods, 10 ml of eachaqueous supernatant was extracted using a C-18 solid-phasecolumn from LiChrolut® (40–63 μm). Metabolites were elut-ed from the column with 2 ml methanol.

Gas Chromatography/Mass Spectrometry

The diketopiperazines (DKPs) were detected and quantifiedby injecting 1 μl of the extract into an Agilent gas chromato-graph 7890A GC System (Waldbronn, Germany) equippedwith a capillary column (Agilent 1901 S-433, 30 m×250 μm×0.25 μm, HP-5 ms column) and a mass selectivedetector 5975C inert XL EI/CI MSD with a quadrupole massspectrometer. The injector was operated at 250 °C and theinjection was pulsed splitless at 10.5 psi for 2 min. Thecolumn temperature started at 60 °C for 5 min, then increased

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from 60 to 300 °C at 20 °C min−1, and was finally maintainedat 300 °C for 5 min to separate substances. The mass spec-trometer conditions were set to 250 °C for the interface, thesource conditions were 230 °C, and the quadrupole tempera-ture was set to 150 °C.

For quantification experiments, standards in three differentconcentrations (0.001, 0.005, 0.01 % (w/v)) were prepared bysolid-phase extraction as described above for the samples. TheGC/MS results of the standards were used to form a calibra-tion curve.

Liquid Chromatography/Mass Spectrometry

The atmospheric pressure ionization (API) mass spectrometryexperiments were carried out using an Agilent Series 1200HPLC system with diode array detector and an Agilent 6120quadrupolemass spectrometer (Waldbronn, Germany). Liquidchromatography was carried out on an endcapped, 5-μm,LiChroCART® 125-4 RP18 column (Merck, Darmstadt, Ger-many) at a flow rate of 0.2 ml min−1. The solvent system usedconsisted of 0.1 % aqueous ammonium formic acid (eluent A)and acetonitrile (eluent B). Elution was done by a gradientwhich ran from an initial value of 100%A for 10min, reached60 % of B within 40 min, increased to 100 % B over 10 min,and maintained 100 % B for a further 10 min. The detectionwavelengths were 220 and 254 nm, and the injection volumewas 5 μl. The MS was used with an electrospray ionization(API-ES) source (dry and nebulizer gas: nitrogen; drying gasflow 10.0 l min−1; nebulizer pressure 45 psig; drying gastemperature 350 °C; capillary voltage 4,000 V). The massspectrometer was used in the positive mode. Settings wereas follows: scan range 40–600m/z, fragmentor voltage 75 V,and gain 1.

Agar Diffusion Assay of Extracted Supernatantand Diketopiperazines

Preparation of the Test Plates for Lysis Activity

Agar diffusion assays were carried out by putting paperdisks (Rotilabo®) loaded with the extracted supernatanton special agar plate media containing bacterial lawns ofthree different species: A. citreus, P. putida, and M. luteus.These were chosen as indicator species because of theirhigh sensitivity to the lysis activity of B. pumilus whichhad been demonstrated in a previous work (Brack et al.2013). Lysis activity against autoclaved, pasteurized, andliving indicators was analyzed. The basal layer of all thelysis test media consisted of a 1.5 % water agar in a 12-mlthin layer in Petri dishes. For media with living indicatorcells, basal agar was supplemented with 0.5 % BactoPeptone (Otto Nordwald, Germany). Microbial host cellsfor lysis indication were cultured in shaking Erlenmeyer

flasks containing nutrient broth II (SIFIN, Berlin) for120 h at 30 °C. Cell suspensions were harvested bycentrifugation for 15 min at 11,000×g at 4 °C. The cellpellets containing the living cells were autoclaved (23 minat 121 °C), pasteurized (30 min at 60 °C), or immediatelyused for preparation of the lysis media. For overlays withpasteurized or autoclaved indicator organisms, a cell con-centration of 0.8 % (w/v) was used. Pellets of living cellswere adjusted to a concentration of 0.4 % (w/v) in a 1 %agar which concerned 0.5 % Bacto Peptone (OttoNordwald, Germany). The mixture was poured on coldbasal agar plates. The agar media holding living bacteriawere incubated for 48 h at 30 °C until a visibly turbidlawn was grown before putting the supernatant-saturateddisks on it. Thus, this lysis test medium contains livinghost cells which were already cultivated to reach a highcell density—unlike the agar diffusion test for antibioticsin which a small inoculum is used to detect growthinhibition.

Extraction of the Supernatants and Dilution of Test/ControlSubstances

The supernatants of B. pumilus SBUG 1921 were extractedaccording to a modified method described by Trindade-Silvaet al. (2009) by lyophilization at −20 °C and 1.030 mbar.Supernatants in 50 ml falcons were extracted using freezedryer ALPHA 1-4, LDC-1 M (CHRIST, Osterode). The dryextracts were frozen at −80 °C.

Dilutions of 1 μg μl−1 in distilled water were prepared fromcommercial cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-AVal-Pro), cyclo(-Leu-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro)from Bachem® as well as a mixture of these six substancesincluding 0.20 μg of each DKP in 1 ml distilled water.Bacitracin (isomers A–F>90 %; Merck) and polymyxin B-sulfate (Calbiochem) were diluted in concentrations of1 μg μl−1 in distilled water as control antibiotics.

Loading and Incubating the Paper Disks

Each disk was loaded with 2 mg of dry extract of B. pumilusSBUG 1921 dissolved in 50 μl methanol. As controls, theantibiotic solutions of bacitracin and polymyxin B and alsothe pure solvent methanol were loaded as 50 μl aliquots ondisks. In the same way, disks were loaded with 50 μl ofaqueous dilutions of the six DKPs. The loaded disks weredried under sterile conditions. Dry disks were put inverselyonto the bacterial lawns of test plates which were kept at 7 °Cfor 4 h to allow diffusion of the substances into the agar media.After 24 h of incubation at 30 °C, newly formed lysis zonesaround the disks indicated bacteriolytic competency. Eachextract and each DKP solution was tested against each indi-cator species in three independent experiments.

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Antibacterial Activity Tests on Microplates

All bacterial test cultures were pregrown by shaking at 30 °Covernight in 500 ml Erlenmeyer flasks containing 50 ml nu-trient broth. As an exception, Bacto Marine Broth (DifcoTM,Otto Nordwald, Hamburg) was used to cultivate halophilicV. ordalii. The precultures were centrifuged gently at 3,800×gfor 8 min at 4 °C, and the cell pellets were suspended withfresh nutrient broth to give a final optical density of 0.2 at awave length of 500 nm (OD500=0.2). One hundred eightymicroliters of cell suspension was added into wells of sterile96-well microplates (Greiner bio-one®). To determine lysis orgrowth inhibition, each well was mixed with 20 μl of aqueousserial dilutions of the DKP stock solution to yield final con-centrations of 100, 50, 25, 12.5, and 1 μg ml−1. Cyclo(-L-Gly-L-Pro), cyclo(-L-Phe-L-Pro), cyclo(-L-Leu-L-Pro), cyclo(-D-Ala-L-Pro), cyclo(-L-Val-L-Pro), and cyclo(-L-Met-L-Pro)from Bachem® served as test compounds for antibacterialactivity tests. As a control, cell suspensions were mixed with20μl of double distilled water. The plates were incubated withshaking at 130 rpm at 30 °C for 24 h. The antibacterial activitywas measured photometrically at 500 nm. For each concen-tration, six growth experiments were performed in duplicateon different microplates.

The inhibition kinetics of the substances on the growth ofA. citreus andM. luteuswas studied over 24 h using a SynergyMx 267118 Monochromator-Based Multi-Mode MicroplateReader and Gen5 Data Analysis Software. Protocol settingswere chosen as follows: temperature 30 °C, wavelengths 500,runtime 24 h, and interval 30 min (49 reads).

Results

Detection and Quantification of Metabolitesby Chromatography and Mass Spectrometry Analysis

As described previously, B. pumilus is able to degrade bothliving and dead cells (Brack et al. 2013). In order to under-stand the mechanism behind this capability, an isolate ofB. pumilus from the Baltic Sea (SBUG 1800) and a laboratorystrain (SBUG 1921) were grown in liquid medium in thepresence of A. citreus cells (LM). Control cultures were grownon a medium supplemented with glucose instead of cells(LCM). Cultures were harvested by centrifugation at the endof the exponential growth phase after 4 h and during thestationary phase at 20 h. The supernatants were extracted bysolid-phase absorption for LC/MS and GC/MS analyses. GC/MS and LC/MS analyses focused on the detection of poten-tially bioactive metabolites in the supernatants. After 4 h ofincubation, GC/MS analysis demonstrated a range of productsat different retention times from 12.5 to 16.5 min (Fig. 1a).

All eluted peaks were examined by comparing them to theNational Institute of Standards and Technology NIST08 massspectral library and commercial standard substances fromBachem®. The six peaks at Rf 13.03, 13.27, 13.60, 14.18,15.67, and 16.29 min were identified as cyclo(-Ala-Pro),cyclo(-Gly-Pro), cyclo(-Val-Pro), cyclo(-Leu-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro). Seven further peaks wereidentified as DKP-like compounds because of similar frag-mentation patterns of the MS spectra to those of the identifiedDKPs (Table 1).

Compounds 1 and 2 with Rf 14.10 and 14.27 min wereidentified by comparing our GC–electron ionization (EI)/MSdata with the results of Ginz and Engelhardt (2001). Theretention time windows of cyclo(-Leu-Pro), cyclo(-Ile-Pro),and cyclo(-Pro-Pro) and isomeric compounds of cyclo(-Leu-Pro) and cyclo(-Ile-Pro) were very narrow for the method usedby Ginz and Engelhardt (2001). The retention pattern [cyclo(-Ile-Pro), cyclo(-Leu-Pro), isomers of cyclo(-Ile-Pro) and cy-clo(-Leu-Pro), and cyclo(-Pro-Pro)] is similar to our GC re-tention pattern, and we reproduced the results of Ginz andEngelhardt (2001) for cyclo(-Leu-Pro). Furthermore, the EI/MS data of cyclo(-Ile-Pro) of Ginz and Engelhardt (2001)with 154 (95), 125 (22), 96 (4), 86 (11), 70 (100), 55 (17),43 (10), and 41 (5) are similar to our results for compound 1(Table 1). The comparison of the Rf and the EI/MS data resultsin the identification of compound 1 as cyclo(-Ile-Pro). Com-pound 2, with Rf 14.27 min, was also identified by comparingEI/MS data to the results of Ginz and Engelhardt (2001) and tothe EI/MS data of Chen et al. (2009) and of Seifert and Merz(2003). The EI/MS data of cyclo(-Pro-Pro) of Ginz andEngelhardt (2001) with 194 (22, M+), 166 (7), 138 (6), 124(6), 110 (14), 96 (15), 70 (100), 55 (9), and 42 (17) and ofChen et al. (2009) with 194M (32), 166 (5), 153 (1), 138 (11),124 (8), 110 (9), 96 (18), 83 (4), 70 (100), 55 (9), and 41 (29),and the figure of the EI/MS spectrum of cyclo(-Pro-Pro) ofSeifert and Merz (2003; data not shown) are very close to ourEI/MS data of compound 2 (Table 1), and we therefore iden-tify this substance as cyclo(-Pro-Pro).

Compound 3 with Rf 15.45 min was identified by compar-ing EI/MS data to the results described by of Seifert and Merz(2003). The EI/MS spectrum of cyclo(-HyP-Pro) shown inWissenschaftliche Berichte FZKA 6944 (mass fragmentationat m/z=154, 41, 83, 111, 70, 43) are very close to our EI/MSdata (Table 1) of compound 3. Furthermore, the fragmentationpattern of cyclo(-Pro-Pro) and cyclo(-HyP-Pro) are similarand we therefore identify this substance with Rf 15.45 minas cyclo(-HyP-Pro).

The mass spectra of all identified DKPs and the com-pounds described as isomers in Table 1 show a base ordiagnostic peak at 70. The peak at 70 indicates a positivepyrrolidine ring structure, a common feature of DKPs. Be-cause of their similarity to the diagnostic peaks in massspectra, similar Rf values, and nearly identical fragmentation

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patterns with the standard substances, the compounds with Rf13.16, 13.73, 14.21, and 16.13 min are tentatively assigned ascyclo(-Ala-Pro), cyclo(-Val-Pro), cyclo(-Leu-Pro), and cy-clo(-Phe-Pro). The differences of retention times may be dueto stereospecific characteristics. The standardDKPs usedwerecyclo(-D-Ala-L-Pro), cyclo(-L-Val-L-Pro), cyclo(-L-Leu-L-Pro), and cyclo(-L-Phe-L-Pro). It may be that different stereo-isomers of these DKPs are secreted by B. pumilus.

The substance with Rf 13.03 min was identified by com-paring the EI/MS data of the commercial standard substancecyclo(-Ala-Pro), and the data agreed well with that of Ginzand Engelhardt (2001) and Wang et al. (2010). The mass

spectrum of cylco(-Ala-Val) published by Wang et al. (2010)corresponds precisely to our data (our MS data in Table 1; 70(100), 44 (61), 168 (56,) 69 (44), 97 (38), and 125 (33); Wanget al. 2010), but it is not identical to the mass spectrum ofcyclo(-Ala-Val) in the standard library they used. The sub-stance which Wang et al. (2010) found in the supernatant ofBurkholderia cepacia is the same as that detected in thesupernatant of B. pumilus, though this cannot be cylco(-Ala-Val). Ginz and Engelhardt (2001) used GC-EI/MS analysisand published a mass spectrum for cyclo(-Ala-Pro) whichdeviates slightly from our mass spectrum data for the com-pound eluting with Rf 13.03 min. Retention time and mass

Fig. 1 a, bGC/MS separation of the 13 identified diketopiperazines (TICshown). a Extracted supernatant of B. pumilus SBUG 1921 after 4 h ofgrowth in LCM; b extracted supernatant of B. pumilus SBUG 1921 after4 h of growth in LM; 1: cyclo(-Ala-Pro), 2: isomer cyclo(-Ala-Pro), 3:

cyclo(-Gly-Pro), 4: cyclo(-Val-Pro), 5: isomer cyclo(-Val-Pro), 6: cyclo(-Ile-Pro), 7: cyclo(-Leu-Pro), 8: isomer cyclo(-Leu-Pro), 9: cyclo(-Pro-Pro), 10: cyclo(-HyP-Pro), 11: cyclo(-Met-Pro), 12: cyclo(-Phe-Pro), 13:isomer cyclo(-Phe-Pro)

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Table1

Chrom

atographicandmassspectroscopicdataforcyclo(-Ala-Pro)and

isom

erof

cyclo(-A

la-Pro),cyclo(-G

ly-Pro),cyclo(-Val-Pro)and

isom

erof

cyclo(-Val-Pro),cyclo(-Ile-Pro),cyclo(-Leu-Pro)

andisom

erofcyclo(-Leu-Pro),cyclo(-Pro-Pro),cyclo(-H

yP-Pro),cyclo(-M

et-Pro),andcyclo(-Phe-Pro)and

isom

erof

cyclo(-Phe-Pro)intheextractedsupernatantofB

.pum

ilusS

BUG1921

aftergrowthin

LM

Com

pound

GC/M

SLC/M

S

Rt(min)

M+

Basepeak

(100)

Diagnostic

peaks

Rt(min)

[M+H]+

[M+Na]+

Standard

Cyclo(-Ala-Pro)

13.03

168

70168(60)

97(41)

44(39)

125(36)

69(35)

24.7

169

B.pum

ilus

Cyclo(-Ala-Pro)

13.03

168

70168(56)

97(38)

44(61)

125(33)

69(44)

24.7

169

Isom

ercyclo(-A

la-Pro)

13.16

168

70168(56)

97(33)

44(36)

125(41)

69(38)

––

Standard

Cyclo(-Gly-Pro)

13.28

154

111

83(95)

154(81)

70(62)

69(47)

41(40)

68(34)

55(32)

98(30)

24.1

155

B.pum

ilus

Cyclo(-Gly-Pro)

13.27

154

111

83(97)

154(81)

70(64)

69(49)

41(44)

68(36)

55(35)

98(32)

24.1

155

Standard

Cyclo(-Val-Pro)

13.60

196

154

70(91)

72(41)

125(40)

41(16)

69(16)

55(11)

68(11)

26.6

197

B.pum

ilus

Cyclo(-Val-Pro)

13.60

196

154

70(83)

72(38)

125(33)

41(15)

69(16)

55(11)

68(10)

135(12)

26.9

197

Isom

ercyclo(-Val-Pro)

13.73

196

154

70(92)

72(21)

125(47)

41(18)

69(14)

55(10)

68(11)

––

Cyclo(-Ile-Pro)/com

pound1

14.10

210

154

70(55)

86(20)

125(13)

41(11)

69(10)

––

Standard

Cyclo(-Leu-Pro)

14.18

210

154

70(59)

86(20)

125(10)

41(9)

69(8)

155(9)

124(9)

28.0

211

B.pum

ilus

Cyclo(-Leu-Pro)

14.18

210

154

70(68)

86(24)

125(10)

41(12)

69(9)

28.0

211

Isom

ercyclo(-Leu-Pro)

14.21

210

154

70(59)

86(16)

125(17)

41(13

69(12)

––

Cyclo(-Pro-Pro)/compound2

14.27

194

70194(41)

96(18)

41(17)

69(14)

68(13)

––

Cyclo(-HyP-Pro)/compound3

15.45

210

7086

(57)

210(49)

124(38)

41(22)

96(20)

68(19)

55(18)

––

Standard

Cyclo(-Met-Pro)

15.67

228

154

70(77)

167(33)

228(33)

139(24)

61(15)

41(11)

56(10)

153(10)

26.9

229

B.pum

ilus

Cyclo(-Met-Pro)

15.67

228

154

70(91)

168(38)

228(26)

139(24)

61(20)

41(11)

56(11)

55(16)

––

Standard

Cyclo(-Phe-Pro)

16.28

244

125

91(46)

70(45)

244(36)

153(35)

120(14)

92(11)

41(8)

28.3

245

B.pum

ilus

Cyclo(-Phe-Pro)

16.29

244

125

91(50)

70(53)

153(35)

120(15)

92(11)

41(10)

28.3

245

267

Isom

ercyclo(-Phe-Pro)

16.13

244

125

91(52)

70(57)

153(37)

120(9)

92(6)

41(7)

––

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spectrum of our compound cyclo(-Ala-Pro), the cyclo(-Ala-Pro) of Ginz and Engelhardt (2001), and the cyclo(-Ala-Val)of Wang et al. (2010) were the same as for the commercialstandard cyclo(-Ala-Pro). On this basis, we identify the sub-stance we elute with an Rf 13.03 min and the substance whichWang et al. (2010) found in the supernatant of B. cepacia ascyclo(-Ala-Pro).

LC/MS analyses accord with GC/MS results concerningthese identified DKPs (Table 1). The DKPs were analyzed byLC/MS only by searching for the molecular mass.

Gas chromatograms indicated that the abundance of DKPsis higher after 4 h of growth of B. pumilus in lysis mediumcompared to growth in the control medium (Fig. 1a, b). Weused commercial standards to create a calibration graph for theexact quantification of the identified DKPs on the basis of GC/MS data (Table 2). Quantification results show that cyclo(-Ala-Pro), cyclo(-Gly-Pro), and cyclo(-Val-Pro) are producedincreasingly after 4 h by both of the test strains. For example,the averaged secretion of cyclo(-Gly-Pro) by B. pumilusSBUG 1921 triples after 4 h from 11.3 μg ml−1 in LCM to34.6 μg ml−1 in LM. B. pumilus SBUG 1800 produces6.7 μg ml−1 cyclo(-Gly-Pro) after 4 h of growth in LCM and26.5 μg ml−1 in LM. The production of cyclo(-Leu-Pro) andcyclo(-Phe-Pro) and the very low concentration of cyclo(-Met-Pro) do not vary markedly between the two media, andby 20 h, the concentrations of these DKPs in the differentculture media are similar (Table 2).

Agar Diffusion Assay

Agar diffusion assays of cell-free supernatant extracts fromB. pumilusSBUG 1921 grown in cell containing lysis mediumand of the standard DKPs were performed in order to inves-tigate the bacteriolytic mechanism. As controls, two sub-stances with bacteriolytic effect on living bacteria were cho-sen, bacitracin and polymyxin B. Bacitracin only lysed livingcells of M. luteus, whereas polymyxin B lysed all living andpasteurized test strains (A. citreus,M. luteus, and P. putida). Incontrast to these control antibiotics, the supernatant extractsalso lysed autoclaved bacterial cells. Supernatant extracts ofLM (20 h) caused lysis zones on lawns of living P. putida andon all of the autoclaved test bacteria, and again the largest lysiszones were seen on autoclaved M. luteus. Extracted superna-tant of LM (4 h) of B. pumilus SBUG 1921 produced smallgrowth inhibition zones on lawns of living M. luteus, but nolysis activity was detected. The lysis control medium LCM(4 h) and (20 h) supernatant extracts and all of thediketopiperazine solutions showed no bacteriolytic activity,and in contrast to the antibacterial activity assays on micro-plates, no inhibitory effect was detected.

Antibacterial Activity Tests on Microplates

Polymyxin B-sulfate (100 μg ml−1) caused decreasing OD500

of the test bacteria species B. pumilus SBUG 1800, B. subtilis

Table 2 Quantification data for the six commercially available DKPsproduced by B. pumilusSBUG 1921 and SBUG 1800 after 4 and 20 h ofgrowth in mineral salt medium containing pasteurized cells of A. citreus

(DKPLM) and mineral salt medium holding 0.01 % glucose instead ofbacterial cells (DKPLCM)

Time Compound Bacillus pumilus SBUG 1921 Bacillus pumilus SBUG 1800

DKPLCM[μg ml−1]

DKPLM[μg ml−1]

Ratio DKPLM/DKPLCM

aDKPLCM[μg ml−1]

DKPLM[μg ml−1]

Ratio DKPLM/DKPLCM

a

4 h Cyclo(-Ala-Pro) 1.2 2.2 1.8 0.5 1.0 2.0

Cyclo(-Gly-Pro) 11.3 34.6 3.1 6.7 26.5 4.0

Cyclo(-Val-Pro) 4.0 7.7 1.9 4.0 6.3 1.6

Cyclo(-Leu-Pro) 2.5 3.0 1.2 2.9 2.7 0.9

Cyclo(-Met-Pro) <0.1 <0.1 0.0 <0.1 <0.1 0.0

Cyclo(-Phe-Pro) 5.3 6.2 1.2 6.8 6.0 0.9

Total 24.3 53.7 2.2 20.9 42.5 2.0

20 h Cyclo(-Ala-Pro) 1.0 1.8 1.8 1.3 0.7 0.5

Cyclo(-Gly-Pro) 22.5 30.4 1.4 16.3 12.8 0.8

Cyclo(-Val-Pro) 3.7 3.9 1.1 4.4 2.9 0.7

Cyclo(-Leu-Pro) 2.6 2.5 1.0 2.8 2.0 0.7

Cyclo(-Met-Pro) <0.1 <0.1 0.0 <0.1 <0.1 0.0

Cyclo(-Phe-Pro) 5.5 5.4 1.0 5.5 5.3 1.0

Total 35.3 44.0 1.2 30.3 23.7 0.8

a >1: increase; <1: decrease; 0: consistent

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subsp. spizizenii, M. luteus, S. aureus, and P. aeruginosa. Theremaining test bacteria species were not lysed, but theirgrowth was inhibited at this antibiotic concentration. The celldensities of these test bacteria increased as the concentrationof polymyxin B-sulfate was reduced.

The tested diketopiperazines did not cause lysis of livingbacteria in microplate assays, but they induced growth inhibi-tion of S. aureus, M. luteus, B. subtilis subsp. spizizenii, andA. citreus at concentrations of 100, 50, and 25 μg ml−1

(Fig. 2a). Using concentrations of 100 μg ml−1 cell, thedensity of these four bacteria lay between 25.7 % of thecontrol value for S. aureus growing with cyclo(-Met-Pro)and 78.7 % for S. aureus growing with cyclo(-Ala-Pro).

Figure 2b illustrates the concentration dependence ofgrowth inhibition in detail at concentrations of 100, 50, 25,12.5, and 1 μg ml−1. Growth-inhibiting effects often remainedconstant even at the lowest DKP concentration of 1 μg ml−1.Experiments on growth kinetics over 24 h on A. citreus andM. luteus appraised by Gen5 Data Analysis Software showedthat growth does not abruptly stagnate after a fixed time orfixed number of cell divisions. Inhibition seems to occurcontinuously over 24 h.

In addition to growth inhibition, growth-promoting effectswere observed for some Gram-positive strains at DKP con-centrations of 12.5 and 1 μg ml−1. B. subtilis subsp. spizizeniiand M. luteus reached significantly higher OD500 at lowconcentrations of cyclo(-Gly-Pro), and low concentrations ofcyclo(-Ala-Pro) promoted growth of both S. aureus andM. luteus.

The Gram-negative species V. ordalii reached higher OD500

in the presence of most DKPs when used separately in con-centrations of 100 μg ml−1. Exceptionally high growth en-hancement of V. ordalii was observed when cyclo(-Gly-Pro)was added. Growth-promoting effects on V. ordaliiwere evendetected at 1 μg ml−1. None of the Gram-negative test bacteriawere inhibited strongly (data not shown).

Discussion

DKPs are cyclic peptides that are derived from two α-aminoacids forming six-membered rings containing two cis-peptidebonds (Fischer 2003). These dipeptides have been shown tobe taste-modulating compounds in food (Ryan et al. 2009) andto facilitate memory in rat brains (Gudasheva et al. 1996). A

few DKPs crosstalk with the LuxR-mediated AHL quorum-sensing system of particular bacteria (Degrassi et al. 2002; deRosa et al. 2003; Holden et al. 1999) and many DKPs showantibiotic activity (de Carvalho and Abraham 2012; Rhee2002). Most Gram-negative bacteria, but also Gram-positivebacteria, fungi, and higher organisms are able to produceDKPs (de Carvalho and Abraham 2012; Ryan et al. 2009).B. subtilis (Lu et al. 2009; Wang et al. 2010) and severalunidentified Bacillus species (de Rosa et al. 2003; Kumaret al. 2013) were found to produce up to ten different DKPs.We have studied the secretion of DKPs by two strains ofB. pumilus from completely different environments usingGC/MS and LC/MS analyses. The spectrum of differentDKPs was surprisingly high compared to that found in otherstudies where normally two to six DKPs are produced (Kumaret al. 2013; Wang et al. 2010). B. pumilus SBUG 1800 andSBUG 1921 secreted 13 proline containing DKPs which wereidentified as cyclo(-Ala-Pro) and isomer of cyclo(-Ala-Pro),cyclo(-Gly-Pro), cyclo(-Val-Pro) and isomer of cyclo(-Val-Pro), cyclo(-Ile-Pro), cyclo(-Leu-Pro) and isomer of cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(-HyP-Pro), cyclo(-Met-Pro),and cyclo(-Phe-Pro) and isomer of cyclo(-Phe-Pro).

To determine the mechanism bywhichB. pumilusdegradesboth living and dead cells, the strains of B. pumilus SBUG1800 and SBUG 1921 were grown in liquid medium in thepresence of pasteurized A. citreus cells. Under these condi-tions, the total DKP concentration doubles after 4 h comparedto the control grown without target cells. For cyclo(-Gly-Pro)which is peak number 3 in Fig. 1a, b, the increase was evenmore marked and reached values of three- to fourfold within a4-h incubation period. These observations are in line with thestudy of Trischman et al. (2004) which describes an enhancedsecretion of cyclo(-Phe-Pro) and indole by a marine Bacillusisolate after a challenge coculture with a different Bacillustarget strain. In this case, the effect was considered to be anexample of competitive induction. In contrast, in our study, wedid not use a closely related strain as challenge to enhanceDKP secretion, but rather pasteurized cells of A. citreus.

We tested the commercially available DKPs against differ-ent Gram-positive and Gram-negative bacteria to examinetheir influence on bacteriolysis and to determine their potentialapplicability as antimicrobial agents. We did not aim to esti-mate the minimum inhibitory concentration (MIC) of DKPs,but rather concentrated on determining their bioactive effectson pregrown cultures and their capacity to inhibit the growthof bacterial lawns. Our results show a distinct growth inhibi-tion effect. However, further work using MIC test conditionswill be needed to determine the maximal inhibition effects.Under these conditions, Qi et al. (2009) achieved considerableinhibition for example of cyclo(-Pro-Phe) against M. luteus.

Our approaches show that the six tested DKPs did notcause lysis of bacterial cells, but did inhibit growth of allGram-positive test strains, except of B. subtilis subsp. subtilis

�Fig. 2 a, b Antibacterial activity of (cyclo(-Ala-Pro), cyclo(-Leu-Pro),cyclo(-Phe-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro), and cyclo(-Met-Pro)separate and as a mixture, indicated by percentage optical density at500 nm (OD500) after 24 h of incubation compared to OD500 reached inDKP-free control medium. aUsing Gram-positive test strains and 100 μgDKP ml−1; b at DKP concentrations of 100, 50, 25, 12.5, and 1 μg ml−1

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DSM 10 Tand DKP producing B. pumilusSBUG 1800. SinceDKPs did not inhibit the growth of similar or closely relatedbacterial strains, the test substances cannot be classified asbacteriocins, but rather as mildly antibiotic substances whichcan reach an active level at comparatively low concentrations(Trischman et al. 2004). The minimal concentration forgrowth inhibition in our tests generally lay between 1 and25 μg ml−1 which is the same range as the values shown incomparable studies (Kumar et al. 2013). Quantification datashow that secretion usually leads to the accumulation ofbioactive DKP concentrations in culture supernatants(Table 2). Only the concentration of cyclo(-Met-Pro)remained at a very low level.

Surprisingly, in our microplate assays, only the Gram-positive test bacteria were growth-inhibited. However, in oth-er studies, DKPs, including several of those tested in thisstudy, do not only inhibit Gram-positives, but also Gram-negative bacteria such as Loktanella hongkongensis, Ruegeriasp. (Qi et al. 2009), E. coli (Wang et al. 2010), and also fungisuch as Candida albicans and Pyricularia oryzae (Kumaret al. 2013; Rhee 2002).

In some cases of growth, clear growth promotion was seenat low DKP concentrations of Gram-positive test strains and atall concentrations using V. ordalii (Fig. 2b). Indeed, the actionof DKPs on Vibrio species seems to be anomalous and mayinvolve mechanisms distinct from those operating in otherbacteria. Indeed, in Vibrio fischeri, DKPs may act as lumines-cence inhibitors antagonizing the LuxR regulon, and inV. cholerae, cyclo(-Phe-Pro) inhibits cholera toxin and toxin-coregulated pilus production by antagonizing the ToxRregulon (Bina and Bina 2010). DKPs may crosstalk also toquorum-sensing systems of Vibrio spp. and in lower concen-trations to the quorum-sensing systems ofM. luteus, B. subtilissubsp. spizizenii, and S. aureus.

Agar diffusion assays on extracted supernatants indicatethe presence of at least one bacteriolytic agent in the superna-tants of B. pumilus grown for 20 h under lysing conditions.These lytic agents could be enzymes or specific antibioticswhich lyse living and autoclaved test bacterial cells. This is incontrast to the antibiotics bacitracin and polymyxin B whichonly caused lysis of living cell lawns.

Bacillus species are able to produce a number of antibioticswhich are mainly produced during the sporulation phase. Forexample, bacitracin is a polypeptide antibiotic produced byBacillus species (e.g., B. subtilis, B. pumilus) inhibiting cellwall biosynthesis in Gram-positive bacteria. This is in accor-dance with our observations that living cells of M. luteus arelysed by the addition of bacitracin. Polymyxin B is anotherantibiotic produced by Paenibacillus polymyxa. This polarpolypeptide antibiotic preferably permeabilizes the outermembrane of Gram-negative bacteria. In our study, bacitracinand polymyxin B caused lysis zones on living and pasteurizedlawns of all three test bacteria, including the Gram-positive

M. luteus and A. citreus. We propose that lysis zones on livingcell lawns induced by the reference antibiotics appear afterenzymatic degradation by the living test bacteria digesting thecell matter disrupted by the antibiotic. On autoclaved lawns,these antibiotics do not cause lysis zones because there are noliving degraders or corresponding thermostable enzymes.However, it remains unclear by which mechanism the agentsin the extracted supernatant cause visible lysis zones onautoclaved cell lawns. Most likely, detected bacteriolytic ac-tivity during agar diffusion assays is not due to extracellularenzymes because the lyophilized supernatants were dissolvedin 100 % methanol in which sparingly water-soluble metabo-lites were dissolved. Concentrated methanol is in general not asuitable solvent to maintain enzyme activity.

In conclusion, this study demonstrates a complex andmultifactorial process of bacteriolysis in our model experi-mental system. Some former publications on B. pumilus ex-plain its antagonistic antifungal activity as being exclusivelydue to the production of the biosurfactant pumilacidin, thoughappropriate tests of the pure substance were not included (deMelo et al. 2009). However, many substances secreted into themedium may contribute to inhibition. Thus, stable exoen-zymes and biosurfactants like pumilacidin may also contributeto the effects seen induced by diketopiperazines. Our studydoes, however, show that bioactive DKPs are excreted underlysis conditions in the late exponential growth phase and ontheir own show growth-inhibiting effects. The mechanism ofinduction remains to be established though it cannot be ex-cluded that the higher production of DKPs in the complexcell-containing lysis medium is a corollary of increased pro-tein degradation. The mechanism of lysis of the targets alsowarrants further investigation.

Acknowledgments The authors thank Anne Reinhard, Stefan Bock,and David Sengebusch for laboratory assistance; Bob Jack for reviewingthe manuscript; Dr. Peter Schumann for the identification of Bacillussubtilis subspecies; and Prof. Dr. Lindequist and Prof. Dr. Riedel forproviding the spectrometers. We also thank the government ofMecklenburg-Vorpommern (Germany) for financial support in the formof a Landesgraduiertenstipendium.

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