Biofilms, the preferred bacterial mode of living and survival, are employed by most microorganisms—which tend to attach to surfaces—to gain physical support, increase nutrient utilization and availability, and augment their resistance against anti-bacterial agents. Rhodococcus ruber (C208) has been shown to form a dense biofilm on polyethylene surfaces while degrading them. Bacterial biofilms comprise bacterial cells embedded in self-secreted extracellular polymeric substances (EPS) whose main components are polysaccharides, proteins and nucleic acids. Revealing the roles of these components will enable further insight into biofilm development and, therefore, the EPS structure-function relationship. The current study focuses on contribution of extracellular DNA to biofilm formation and stability. This was approached by investigating the influence of nucleases on biofilm formation via degradation of their corresponding substrates within the biofilm of C208. RNase application to cultures of C208 decreased biofilm formation. Degradation of biofilm DNA by DNase reduced early-stage biofilm formation by 20% -25% but had no significant effect on established, mature biofilm. Likewise, the addition of DNA to cultures significantly enhanced early-stage biofilm formation by 50% -100%. RAPD-PCR analysis revealed different band patterns from intra-cellular DNA and extra-cellular DNA and also between the supernatant and biofilm fractions of extra-cellular DNA, indicating that perhaps only certain DNA molecules are utilized as part of the biofilm.
Polyethylene (PE), the most commonly used synthetic polymer, is highly inert and virtually non-biodegradable. Current global production of PE stands at about 140 million tons per year. In the absence of safe disposal procedures, plastic waste accumulates in the environment posing an ever increasing ecological threat to terrestrial and marine wild life [
Biodegradation has been suggested as the best approach for degrading polyethylene (Reviewed by Sivan [
After initial biofilm establishment, cell-to-cell communication (i.e., quorum sensing) via extracellular signaling molecules regulates further modification and development of the biofilm [
The preferred mode of growth of plastic-degrading bacteria seems to be based on the formation of a biofilm on the plastic surface. Since plastic polymers such as polyethylene and polystyrene are hydrophobic, the bacterial surface must be also hydrophobic to form a stable biofilm [
Recently, we isolated a strain of Rhodococcus ruber (designated C208) that utilizes polyethylene as a sole carbon and energy source and that degrades the polymer. This Gram-positive bacterium is an actinomycete that showed a high level of hydrophobicity [
Bacterial biofilms are encased within an extracellular matrix consisting of polysaccharides, proteins and nucleic acids [
Gram-positive bacteria release eDNA to the biofilm, apparently by autolysins (bacterial murein hydrolases). These molecules were reported to be involved in biofilm formation, mediating bacterial lysis in Enterococcus faecalis and Staphylococcus epidermidis [23,27,29,30].
Here we further assess the role of Rhodococcus ruber C208 eDNA in biofilm formation and stability.
Cultures of Rhodococcus ruber C208 were cultured in 100 ml nutrient broth (NB; Difco), incubated in flasks (250 ml) on a rotary shaker (120 rpm) at 30˚C or on nutrient agar (Difco) Petri dishes at 30˚C. Cultures of Bacillus cereus and Pseudomonas aeruginosa from our laboratory collection were maintained in 100 ml Luria Broth (LB) and incubated as above or on LB agar Petri dishes as above.
Experiments were performed in a minimal salts medium (SM; [
Aliquots from diluted cultures were transferred to PS microplates (190 μl per well) and incubated for the desired time at 30˚C. When required, DNase I (RNase-free) or RNase A (DNase and protease-free) (Fermentas, Vilnius, Lithuania) was added to the wells, at time of seeding or after 24 h of growth, to achieve a final concentration of 15 U/ml or 50 mg/ml, respectively. DNase I heatinactivated samples (15-min incubation at 65˚C) served as control. Prior to processing, the turbidity of the culture in the wells was determined at 595 nm with a plate reader (Biotek ELx808, Winooski, VT, USA) and serial diluted for CFU counts.
The metabolic activity of the bacterial biofilm was determined by metabolism monitoring with CTCFluorescent Redox Probe (monitoring reduction of CTC (5- cyano-2,3-ditolyl tetrazolium chloride) to CTCF (Formazan violet) by bacterial respiration. [31,32].
Treated and untreated cultures grown in the PS microplates were decanted and the wells were washed twice with distilled water and air-dried. Afterward, 200 μl crystal violet (0.2% w/v) (Sigma-Aldrich) were added to the wells, which were incubated for 15 min and then washed twice with distilled water to remove loosely attached biomass, and the wells were air-dried again. Crystal violet retained by the cells was subsequently re-dissolved in 200 μl ethanol (99% w/v), and after incubation of 1 h, absorbance was determined with the plate reader at 595 nm [
Intra-cellular DNA was extracted from 24-h liquid cultures in SM supplemented with polyethylene films using the UltraCleanTM Microbial DNA isolation kit (MO BIO, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two fractions of eDNA were extracted: S— supernatant DNA from cell-free supernatants [by centrifugation at 10,000 × g for 10 min at 4˚C and filtration (0.2 μm)] of cultures grown in liquid SM medium supplemented with polyethylene films, and B—biofilm DNA obtained from the washed biofilms adhered to the surface of the polyethylene films. The biofilm was gently scraped, re-suspended in sterile deionized water and vortexed. The suspension was then centrifuged (10,000 × g for 2 min at 4˚C) and filtered, as above. This resulted in a cell-free fraction for DNA isolation.
Short fragments of C208 genomic DNA were produced by the amplification of two 16S rRNA gene fragments. The resultant amplicons were confirmed by agarose gel electrophoresis to be about 300 bp and 500 bp (forward primer 8F, reverse primers 341R and 518R, respectively). The DNA fragments were added to the growth medium at increasing concentrations, and biofilm formation was compared to that obtained in the presence of intact genomic DNA.
Genomic DNA (3 μg/ml) in Ultrapure® water was used to pre-coat the wells of the PS microplate under incubation for 20 h at 4˚C. The solution was decanted and the wells were air-dried prior to inoculation with the bacteria and assessment of biofilm mass (Modified from [
Profiling of intraand extra-cellular DNA fractions (obtained as detailed above) was performed by RAPD-PCR using ten mer primers. A primer mixture of 208- ACGGCCGACC/228-GCTGGGCCGA was used, Bacillus DNA served as control and gel was run in TAEx1 buffer; Or Primer 241-GCCCGAGCGG was used with Pseudomonas DNA as control and gel run in TBEx1 buffer [35-38].
Extracellular DNA was obtained from two fractions, supernatant DNA (S) and biofilm DNA (B). RAPD-PCR analysis was conducted in an attempt to distinguish between the eDNA sources. The analysis showed different patterns of PCR products between the eDNA fractions and the cellular DNA (C) (
Extracellular DNA found as part of the bacterial EPS is a novel finding and its presence and characterization in Rhodococcus ruber’s biofilm was first investigated in our lab.
To further elucidate the role of eDNA in our strain, we amended cultures of C208 with DNase I and RNase A. All biomass quantifications of the biofilm were conducted by crystal violet (CV) staining as detailed in the Materials and Methods chapter. First we verified that the bacteria were not damaged by the nucleases. Respiration test showed no harm to their viability (
The enzymes were added at inoculation time or to 24-h old biofilms. In
Biomass quantification of the biofilm after 5 days showed that both enzymes reduced its formation without affecting culture growth or vitality (
Addition of Bovine Serum Albumin (BSA), caused a significant increase in growth rate, while almost no biofilm was formed (
bacteria preferred the planktonic mode when food was abundant. Addition of DNase I and RNase A to 24-h old biofilms had very little effect (data not shown). These findings suggest that the nucleic acids are important to the formation of C208 biofilm mainly in the initial stages of its development.
Since eDNA degradation affected the biofilm we wondered if addition of excess DNA molecules would have any influence on it. DNA was extracted from C208 cells (endogenous DNA) and used at a final concentration of 3, 30 or 100 ng/ml. To rule out the effect of self DNA, the same experiments were repeated using salmon sperm DNA (exogenous DNA). As shown in
The addition of DNA to a 2-day-old biofilm enhanced biofilm formation, but cell growth was also improved due to the utilization of the DNA not only as a biofilm support, but also as a nutrient, and the net effect is unequivocal (
To find out whether the size of the DNA molecule is significant for supporting biofilm formation we added short DNA fragments obtained from C208 DNA, as detailed in the Materials and Methods. Our results showed that biofilm development was similar to that of biofilms grown in the absence of additional DNA (Data not shown).
Next, we sought to evaluate whether the DNA-driven enhancement of biofilm formation was apparent only during the initial steps of biofilm establishment (initial cell attachment) or whether the presence of DNA was also required during the subsequent early phases of biofilm development. C208 DNA was used to pre-coat the wells of the PS plates (verified by propidium iodide staining), and the bacteria were incubated in DNA-free medium. The similarity in biofilm masses of the coated well and the uncoated control (
To further understand the contribution of eDNA in biofilm formation, we tried to eliminate the influence of DNase by adding excess DNA to the same growth medium (
Biofilms are highly structured communities of microorganisms attached to a surface. It has been proposed that all bacterial biofilms have a number of functionally conserved components in common, including the production of an extracellular polysaccharide matrix, extracellular DNA and proteins.
The present study shows that DNA contributes to the development of the biofilm of Rhodococcus ruber C208.
Our findings confirm that extracellular DNA (eDNA) is an important structural component of C208 biofilm. Exposure of the biofilm to DNase I resulted in eDNA degradation and a subsequent reduction in biofilm formation. These results are supported by studies in other species where DNA was reported to be an essential component of the extracellular polymeric substances [24,27,39,40]. Whitchurch et al. [
Mulcahy et al. [
This structural role in C208 biofilm is further reinforced by the effect obtained when the bacteria were supplemented with DNA (either endogenous or foreign), which enhanced biofilm formation. It should be noted that the inhibitory effect of DNase I and enhancing effect of additional DNA occurred mainly during the early stage of biofilm development, while during maturation the effect was less obvious.
Incubation of RNase with cultures of C208 demonstrated a decrease similar to that obtained with DNase. Existence of extracellular RNA has been reported by Nishimura et al. [
We also found that DNA is not involved in the initial attachment stage since pre-treatment of the plastic substrate with DNA did not enhance biofilm formation. Similar results were obtained with the Gram-positive bacteria Enterococcus faecalis [
Numerous studies indicate that some bacteria produce substantial quantities of extracellular DNA (eDNA) through mechanisms that are thought to be independent of natural cell death. In some cases, eDNA secretion may be mediated by the positive regulation of proteases, which contribute to biofilm formation via the regulation of active cellular autolysis and DNA release [27,29,30]. Other mechanisms, especially in Gram-positive bacteria, involve murein hydrolases, which were reported to be involved in biofilm formation, mediating bacterial lysis in Enterococcus faecalis and Staphylococcus epidermidis [23,27,29,30,46,47]. Other reports demonstrate them as adhesins that contribute to bacterial attachment to polymeric surfaces and biofilm formation via the release of eDNA [23,48,49]. Elucidation of the role of eDNA in biofilm formation could be advanced by tracking its source. We observed sequence discrepancies between eDNA and cellular DNA in C208 in the RAPD-PCR analysis, a finding that was also confirmed by Nishimura et al. [
The findings indicate that eDNA is an important structural component involved in the biofilm formation of C208 to date, but more research is needed to fully elucidate its role.