Biofilm Formation by Bacillus cereus Is Influenced by PlcR, a Pleiotropic Regulator

The ${Delta}$ plcR mutant of Bacillus cereus strain ATCC 14579 developedsignificantly more biofilm than the wild type and produced increasedamounts of biosurfactant. Biosurfactant production is requiredfor biofilm formation and may be directly or indirectly repressedby PlcR, a pleiotropic regulator. Coating polystyrene plateswith surfactin, a biosurfactant from Bacillus subtilis, rescuedthe deficiency in biofilm formation by the wild type.

INTRODUCTION

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Introduction

Bacillus cereus is a pathogen that causes two distinct typesof food poisoning, the diarrheal and emetic syndromes, as wellas a variety of local and systemic infections such as endophthalmitis,endocarditis, meningitis, periodontitis, osteomyelitis, woundinfections, and septicemia (12, 32). B. cereus can be readilyisolated from food and agricultural products, as well as soil,vegetation, dust, and natural waters. It is regarded as oneof the common organisms that impair the quality of dairy products(23, 25, 30, 34). Its ubiquitous nature, combined with its abilityto sporulate and grow at refrigeration temperature, make itdifficult to control. B. cereus has been shown to be able toform biofilms on plastic, glass wool, and stainless steel (2,26, 28), and the biofilm cells were more resistant than planktoniccells to chemical sanitizers (29). Biofilm accumulation in foodprocessing environments can lead to decreased food quality andsafety (20, 35, 36), which impacts public health as well asthe economy.

In the present study, the role of PlcR in biofilm formationby B. cereus strain ATCC 14579 was investigated. PlcR, a pleiotropicregulator, is activated by a small diffusible peptide (PapR)that acts as a quorum-sensing effector (33). It controls theexpression of a variety of genes, many of which encode potentialvirulence factors, including enterotoxins, hemolysins, phospholipasesC, and proteases (1, 7, 15). We found that biofilm formationwas enhanced under low nutrient conditions and was dependenton biosurfactant production, which was directly or indirectlyrepressed by PlcR.

Biofilm formation.

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Biofilm formation.

B. cereus ATCC 14579 and its ${Delta}$ plcR mutant (31) were grown inLuria-Bertani (LB) broth (Difco/Becton Dickinson, Sparks, Md.)at 32°C and 200 rpm overnight to generate inoculum cultures.For the ${Delta}$ plcR mutant, kanamycin was added at a final concentrationof 150 µg/ml. Since nutrient availability is one of themajor factors affecting biofilm formation, we compared the abilityof the wild-type and mutant strains to develop biofilms in richand low-nutrient media. Overnight cultures were adjusted toan optical density at 620 nm (OD₆₂₀) of 0.01 in LB or EPS, alow nutrient medium that contained 7 g of K₂HPO₄, 3 g of KH₂PO₄,0.1 g of MgSO₄ · 7H₂O, 0.01 g of CaCl₂, 0.001 g of FeSO₄,0.1 g of NaCl, 1 g of glucose, and 0.125 g of yeast extract(Difco) per liter (11). Then, 2 ml was added to wells of polystyrene24-well plates (Falcon/Becton Dickinson, Franklin Lakes, NJ),followed by incubation at 32°C and 50 rpm for 8 h. The totalgrowth (OD₆₂₀) in each well was measured; planktonic bacteriawere removed, and the wells were washed with distilled waterand air dried. Biofilm cells were stained with 2 ml of 0.3%crystal violet for 10 min, washed with distilled water, andair dried. The crystal violet in the biofilm cells was solubilizedwith 2 ml of 70% ethanol, and the optical density at 590 nm(OD₅₉₀) was measured (13). The total growth of the wild typeand the ${Delta}$ plcR mutant was similar in EPS and LB; however, biofilmformation in EPS by the ${Delta}$ plcR mutant was about four times higher(P < 0.05) than that by the wild-type strain (Fig. 1). Adramatic decrease in biofilm development was observed in a richmedium such as LB. Recently, Auger et al. (2) also observedthat LB did not support biofilm formation by B. cereus ATCC14579. We therefore used EPS for subsequent experiments.

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FIG. 1. Biofilm formation in LB and EPS. Total growth was measured as the OD₆₂₀ ( ${square}$ ), and biofilm formation was measured as the OD₅₉₀ ( ${blacksquare}$ ). The data represent the means (± the standard deviations) of 12 wells from two independent experiments.

To monitor biofilm development over time, overnight cultureswere adjusted to an OD₆₂₀ of 0.01 in EPS, and 10 ml was addedto 60-by-15-mm polystyrene petri dishes (Falcon), followed byincubation at 32°C and 50 rpm. At specific times, planktoniccells were removed, and biofilm cells were rinsed and air dried.For enhanced visualization of biofilm cell morphology and structure,cells were fixed with 2 ml of 1% glutaraldehyde, stained with10 ml of acridine orange (0.025% in 0.026 M citric acid buffer[pH 6.6]; Sigma, St. Louis, Mo.), washed, and air dried. Biofilmcells were observed with an Olympus BH-2 microscope equippedfor epifluorescence with an HB0100W mercury burner lamp, a 490-nmexcitation filter, and a 515-nm barrier filter. At 6 h, veryfew cells of the wild type had attached to the bottom of theplate, whereas the ${Delta}$ plcR mutant started to develop a biofilm(Fig. 2). The ${Delta}$ plcR mutant biofilm reached maximum density at12 h, after which the cells started to detach. At 36 h, fewcells remained on the plate. A modest increase in attachmentwas observed for the wild type by 12 h and, as with the mutant,the cells started to detach with prolonged incubation.

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FIG. 2. Time course of biofilm formation. Biofilms were developed for 6, 12, 24, and 36 h; stained with acridine orange; and observed by epifluorescence microscopy (x100 magnification). Bars, 200 µm. The results shown are representative of three independent experiments.

Determination of biosurfactant production.

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Determination of biosurfactant...

In a separate study to examine motility (unpublished data),we observed that when grown on EPS plates containing 0.7% agar,the ${Delta}$ plcR mutant but not the wild type formed dendritic coloniessimilar to those observed in Bacillus subtilis (17). In addition,a delimiting ring was visible around the B. cereus ${Delta}$ plcR mutantcolony but not the wild-type strain at 60 h (data not shown).A delimiting ring became visible around the wild-type colonyat 72 h (Fig. 3A); however, the ring was much smaller than thatof the ${Delta}$ plcR mutant. Delimiting rings were first described byJulkowska et al. (17, 18), who observed that a transparent zone,delimited by a narrow ring, preceded the advancing fronts ofdendritic colonies of B. subtilis. The rings were due to thesecretion of surfactin, a lipopeptide biosurfactant. When adrop (5 µl) containing 0.05 to 50 µg of surfactin(Sigma) was allowed to spread on the EPS agar surface, a delimitingring was visible, and the ring diameter increased with increasingsurfactin concentration. A representative ring formed by 1.25µg of surfactin is shown in Fig. 3A.

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FIG. 3. Formation of delimiting rings on EPS 0.7% agar. (A) Delimiting rings (see arrows) were formed by the wild type and ${Delta}$ plcR mutant at 72 h. A 5-µl (1.25 µg) portion of B. subtilis surfactin was spotted onto the agar surface as a control. The delimiting rings were photographed by reflected light. (B) A 3-µl portion of trypan blue was spotted within the ring formed by the wild type, ${Delta}$ plcR mutant, and surfactin. In the bottom panel, trypan blue was spotted on an uninoculated plate. Bars, 0.5 cm. The results shown are representative of three independent experiments.

Biosurfactant compounds are produced by many bacteria and canhave a variety of structures, such as neutral lipids, phospholipids,glycolipids, and lipopeptides (14). One of their propertiesis the ability to reduce surface tension (4, 27). To visualizeany surface tension change that may be associated with the delimitingring, a drop of trypan blue (3 µl of 1.5 mg/ml) was spottedwithin the delimiting ring and on an uninoculated area as acontrol. The drop spread wider in the ring area of the ${Delta}$ plcRmutant colony and surfactin-coated plate than in the wild type(Fig. 3B). This suggests that the delimiting ring could be dueto biosurfactant production and that the ${Delta}$ plcR mutant producedmore biosurfactant than the wild type. After a BLAST searchof the whole genome sequence of B. cereus ATCC 14579, no srfoperon, which harbors the surfactin synthetase genes, was found.This suggests that the biosurfactant produced by B. cereus ATCC14579 is not surfactin. There are a very limited number of studieson biosurfactant production in B. cereus. A monoglyceride biosurfactant(9) and plipastatin (24), a lipopeptide that potentially mayhave biosurfactant activity, have been reported.

We partially purified biosurfactants from the wild type andits ${Delta}$ plcR mutant and also from B. subtilis strain 3A1 (BacillusGenetic Stock Center; same as ATCC 6051), which produces surfactin.Cultures were grown in 500 ml of EPS and incubated at 32°Cand 50 rpm for 72 h. Biosurfactant was purified as describedby Kim et al. (19) and quantitated by using the modified drop-collapsemethod (5). Biosurfactant concentrations in the samples weredetermined by using a standard curve generated with differentconcentrations of surfactin. A linear correlation was foundbetween surfactin concentration and drop diameter, in the rangeof 0 to 0.0375 mg/ml (r² = 0.912). The ${Delta}$ plcR mutant producedmore biosurfactant (6 ng/ml) than did the wild type (0.5 ng/ml).B. subtilis 3A1 produced the highest amount (40 ng/ml).

Surfactin-coated plates promote biofilm formation.

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Surfactin-coated plates promote...

The higher amount of biosurfactant produced by the ${Delta}$ plcR mutantmay play a role in its increased ability to a biofilm comparedto the wild type. To test this, we used surfactin, which iscommercially available and easily quantified. Portions (2 ml)of increasing amounts of surfactin (0.1 to 1 mg/ml) were addedto 60-by-15-mm polystyrene petri dishes and aspirated after10 min, and the dishes were air dried for 30 min. These surfactin-coateddishes were used to generate biofilms by the wild type as describedabove. For dishes coated with 0.1 mg of surfactin/ml, biofilmformation was evident by 12 h, and the biofilm remained wasrobust up to 24 h, while as noted previously minimal biofilmformed on the uncoated surface (Fig. 4). After 40 h, most ofthe biofilm cells had detached. Similar results were obtainedwhen surfactin concentrations of 0.2 or 0.5 mg/ml were used(data not shown). Biofilms formed by the ${Delta}$ plcR mutant on surfactin-coateddishes (0.1 to 0.5 mg/ml) were more abundant than on uncoateddishes at 12 and 24 h. However, when the concentration was increasedto 1 mg/ml, no biofilm was developed by the wild type or themutant (data not shown). Growth was not affected by any of thesurfactin concentrations we tested. It appears that biofilmformation by B. cereus is supported by an optimal concentrationrange of biosurfactant.

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FIG. 4. Effect of surfactin on biofilm formation. Biofilms were developed for 12, 24, and 40 h; stained with acridine orange; and observed by epifluorescence microscopy (x400 magnification). Petri dishes were precoated with 2 ml of surfactin (0.1 mg/ml; right column) or not precoated (left column) prior to biofilm development. Bars, 200 µm. The results shown are representative of three independent experiments.

Biosurfactants have been reported to play various roles in biofilmformation. Pellicle or surface biofilm formation in B. subtilisalso required surfactin (8). It has been reported that the additionof about 0.1 mg of surfactin/ml could rescue pellicle formationby surfactin-deficient mutants of B. subtilis A1/3 (16). Surfactinproduced by B. subtilis 6051 was required for biofilm formationin microtiter plates and on Arabidopsis root surfaces (3). Incontrast, the addition of surfactin inhibited biofilm formationby Salmonella enterica serovar Typhimurium, Escherichia coli,and Proteus mirabilis but not by Pseudomonas aeruginosa (22).Rhamnolipid, a biosurfactant produced by P. aeruginosa, wasreported to be involved in the development (21) and maintenanceof biofilm architecture (10, 21). However, Boles et al. (6)observed that rhamnolipids mediated the detachment of P. aeruginosafrom biofilms.

In conclusion, biofilm formation by B. cereus ATCC 14579 onpolystyrene is promoted by low-nutrient conditions and is relatedto the production of a biosurfactant, which appears to be negativelyregulated by PlcR. The involvement of a biosurfactant and PlcRin biofilm formation by B. cereus has not been reported previously.All of the genes belonging to the PlcR regulon are positivelyregulated by PlcR, suggesting that the negative role of PlcRon biosurfactant production is likely indirect. Whether biosurfactantbiosynthetic genes are controlled by PlcR or by other genesunder PlcR regulation remains to be determined.

ACKNOWLEDGMENTS

This research was supported by Hatch funds (WIS04799) and bythe College of Agricultural and Life Sciences, University ofWisconsin—Madison.

FOOTNOTES

* Corresponding author. Mailing address: Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, 1925 Willow Dr., Madison, WI 53706. Phone: (608) 263-1168. Fax: (608) 263-1114. E-mail: acwong{at}wisc.edu.

REFERENCES

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