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Applied and Environmental Microbiology, December 2005, p. 8987-8990, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8987-8990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Cell-Cell Influences on Bacterial Community Development in Aquatic Biofilms{dagger}

Robert J. C. McLean,1* Mary B. Barnes,2 M. Katy Windham,3 Mubina Merchant,1,{ddagger} Michael R. J. Forstner,1 and Clay Fuqua4

Department of Biology, Texas State University—San Marcos, 601 University Drive, San Marcos, Texas,1 Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, Louisiana,2 BRB Lab C6, University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, Texas,3 Department of Biology, Indiana University, Jordan Hall, 1001 East 3rd St., Bloomington, Indiana4

Received 1 October 2004/ Accepted 17 August 2005


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ABSTRACT
 
Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.


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INTRODUCTION
 
Bacterialsurface colonization and biofilm formation on rocks and other abiotic substrata have been widely documented in aquatic environments. Although the populations and cell surfaces of planktonic bacteria affect biofilm populations (15, 17), there is growing evidence that interactions among species may also be important. Using karst aquifer isolates, Whiteley et al. (24) showed that an increased diversity of bacterial species enhanced biofilm population density, suggesting the importance of population interactions in the development of aquatic biofilms. Quorum sensing (9) is important in biofilm formation and physiology (7, 8) under some environmental conditions (21). Many gram-negative bacteria use N-acylated homoserine lactones (AHLs) as quorum signal molecules (9). AHLs have been detected within biofilms (20), where they have been shown to be an integral part of biofilm development (8). Several plants have been shown to be able to control biofouling in situ by interfering with quorum sensing (11, 13). Supplementation of biofilms with AHLs has resulted in alterations in physiology (4, 22). In light of this work, we conducted field investigations to see whether two different AHLs, N-octanoyl-L-homoserine lactone (C8-HSL) and N-(3-oxooctanoyl)-L-homoserine lactone (3-O-C8-HSL), could influence recruitment of bacteria to biofilms.


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Bacterial strains and culture media.
 
The bacterial strains and media used are listed in Table 1. AHL production (or lack of production in controls) in all strains was confirmed using a bioassay (20). All organisms were incubated at 30°C.


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  		TABLE 1. List of strains used


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Recruitment assay.
 
Spent culture supernatants (referred to as "baits") were prepared from overnight broth cultures of Agrobacterium tumefaciens strains R10(pCF218) and WCF47(pCF218), a traI mutant of R10 (26), by centrifugation at 10,000 x g for 5 min. Commercially available C8-HSL (Fluka) was added at a final concentration of 1 mg/ml–1 to some of the culture supernatant of strain WCF47(pCF218). The liquid "baits" used were (i) an R10 supernatant (autoclaved), (ii) a WCF47 supernatant (autoclaved), (iii) a WCF47 supernatant plus C8-HSL (autoclaved), (iv) a sterile LB broth, (v) lake water, and (vi) unfilled tubing. Weighted and unweighted clamps were used to seal each end of the tubing. Field studies were performed adjacent to a wooden deck near Spring Lake (29°53'32''N, 97°55'55.7''W) in San Marcos, Texas (16). Duplicate dialysis tubes (500 molecular weight cutoff [MWCO]) were attached to the deck by string so that the tubes were submerged below the water surface and separated by 25 cm. Following a 5-h exposure, the tubing was retrieved and analyzed.

Microbiological (dilution plating) analysis was performed with the strains used to prepare the conditioned media—the tubing contents as well as the bacteria adherent to the tubing. Bacterial adhesion and biofilm formation on the tubing were confirmed by scanning confocal laser microscopy (3). For tubing contents, 1 ml liquid was aspirated and stored at –20°C for analysis by denaturing gradient gel electrophoresis (DGGE). For tubing-adherent (biofilm) bacteria, the tubing was placed in a sterile petri dish and a 2.5-cm2 section removed. The tubing section was then placed in a vial containing 10 ml sterile water, dipped gently to remove nonadherent bacteria, and placed in a second vial with 10 ml sterile water, and adherent bacteria were enumerated by sonication and dilution plating (24). The remaining liquid from the biofilm sonicate (9 ml) was frozen at –20°C for subsequent DGGE analysis. All dilution plating was completed within 3 h of the field tests. Based on colony morphology, 70 representative isolates were subcultured and stored at –80°C (24).


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Bacterial analysis.
 
Representative isolates were retrieved from frozen storage, cultured on R2A agar, and tested for AHL production and potential quorum signal inhibition (19). Representative isolates were analyzed by DGGE and partial (500-bp) sequencing of 16S rRNA genes. Genomic DNA extraction was done using a freeze-thaw technique (12) or a Bactozol DNA isolation kit (Molecular Research Center, Inc., Cincinnati, OH) in accordance with the manufacturer's instructions. For PCR amplification of 16S rRNA genes, the primers used included 27F and 518R for sequencing and GC357F and 518R for sample preparation prior to DGGE analysis (12). A touchdown PCR protocol was used (12), and PCR yields were monitored by agarose gel electrophoresis. Gels were stained with ethidium bromide (0.5 µg/ml) and imaged using a model TFM-26 UV transilluminator (UVP, Inc., Upland, CA) covered with a glass protector (Fotodyne, Hartland, WI) and a Nikon 990 Coolpix digital camera with a UV lens filter (Wratten). Electronic images were optimized for contrast using Adobe Photoshop version 7.0.1. DGGE was performed using the DCode system (Bio-Rad) as described previously by R. J. C. McLean, A. K. Welsh, and T. R. Simpson (submitted for publication). Gel conditions were as follows: 8% acrylamide gel in 0.5x Tris-acetate-EDTA, 30 to 50% urea-formamide denaturant, a running buffer of 0.5x Tris-acetate-EDTA, a running temperature of 60°C, and electrophoresis conditions consisting of 130 V for 3.5 h. In the absence of commercially available DGGE standards, we used a 1:1 mixture of PCR-amplified 16S rRNA genes from Escherichia coli DS291 (3) and Pseudomonas aeruginosa ATCC 10145 as internal standards for each DGGE gel. Standards and samples were loaded (15 µl sample and 15 µl 2x loading dye). DGGE gels were stained and imaged as described above.


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16S rRNA gene sequence-based identification of isolates.
 
Partial sequencing (ca. 500 bp) of the V3 region of 16S rRNA genes was used to identify representative isolates and to provide a comparison to the "bait strains" (strains used to prepare the conditioned medium). While some strains were sequenced in the laboratory, the majority of the strain sequencing was performed by MIDI Labs (Newark, DE) (25). Sequencing information, GenBank accession numbers, and phylogenetic analyses are shown in the supplemental material.


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Influence of conditioned medium and AHLs on biofilm formation.
 
The dialysis tubing placed in Spring Lake for 5 h was colonized by bacteria. One-way analysis of variance showed no significant differences in colonization levels (CFU cm–2) (P = 0.501) among the treatments (Fig. 1). In contrast, the diversity of organisms colonizing the tubing was higher in the abiotic controls (treatments 4 to 6) and was reduced in the presence of conditioned media (treatments 1 to 3) (Fig. 2). Sequencing and phylogenetic analysis confirmed these results (see the supplemental material). Of note, most of the strains that colonized the conditioned media from A. tumefaciens strains were members of {alpha}-Proteobacteria along with two strains of {gamma}-Proteobacteria. Of those {alpha}-Proteobacteria isolates, the majority were related most closely to the genus Agrobacterium or, in one instance (A62-48), to a closely related genus, Sinorhizobium. The two isolates of {gamma}-Proteobacteria (A62-45 and A62-2) might represent novel organisms, as they are not closely related to any known species. The greater diversity of strains colonizing the abiotic control tubing (treatments 4 to 6) included members of the {alpha}-, ß-, and {gamma}-Proteobacteria. Neither gram-positive bacteria nor gram-negative bacterial clades other than the proteobacteria mentioned above were isolated. AHLs had some influence on colonization. There was an indication (Fig. 2) that one DGGE band was present in R10-conditioned medium as well as WCF47-conditioned medium supplemented with C8-HSL. The DGGE band was not present under other conditions, suggesting that at least some organisms may colonize a surface in response to AHLs. Several explanations for this result are possible. First, the bait organisms may need to be living and actively synthesizing AHLs and other metabolites in order to attract other organisms. Second, it is possible that an AHL effect on colonization may not be immediate but may be delayed due to AHL-mediated gene activation (9, 23) in the already colonized ("bait") organism or the incoming organism. As C8-HSL was added immediately prior to field exposure, it is possible that the reduced C8-HSL supplement has little or no effect or at least a delayed effect not seen during the brief exposure to the WCF47(pCF218) strain. C8-HSL with a molecular weight of 227 would diffuse readily from dialysis tubing, so its presence, even in dialysis tubing with a molecular weight cutoff (MWCO) of 500, would be transient. These issues will be explored in future studies. Our observations here are not without precedent. In laboratory experiments, Gilbert et al. (1) showed that supplementation with C6-HSL and Pseudomonas fluorescens spent culture medium enhanced biofilm formation by this same organism. Valle et al. showed that AHLs can influence sludge community composition (22). AHLs have occasionally been used as medium supplements for the culturing of some fastidious organisms (5).



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  		FIG. 1. Dialysis tubing (500-MWCO) colonization experiment showing bacterial colonization after 5 h in the presence of autoclaved conditioned medium from A. tumefaciens R10(pCF218) (AHL supplemented), A. tumefaciens WCF47(pCF218) (AHL nonsupplemented), and A. tumefaciens WCF47(pCF218) supplemented with C8-HSL (+C8). Abiotic controls are also present. Details are given in the text.



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  		FIG. 2. DGGE showing the diversity of bacteria that colonized dialysis tubing (500 MWCO) with autoclaved conditioned medium from A. tumefaciens R10 (wild type) and A. tumefaciens WCF47 ({Delta}traI) with (+C8) and without (no) supplementation with C8-HSL. DNA from the "bait" strains was not detected in conditioned medium that had been autoclaved, whereas there was some indication of colonization in response to AHLs by bacteria on tubing containing autoclaved R10 (AHL supplemented) and C8-HSL-supplemented WCF47 (arrowheads). E. coli (E) and P. aeruginosa (P) standards are also indicated.


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Microbial influence on biofilm diversity.
 
In contrast to biofilm population diversity patterns seen on abiotic control tubing (Fig. 3), reduced population diversity was observed in the presence of conditioned media (Fig. 2) regardless of whether the media contained AHLs. This was the most striking observation in the current study and would imply that several factors in addition to AHLs may influence microbial succession (6) and biofilm community development on abiotic surfaces. Although cell surface interactions (14) and changes in chemical and physical microenvironments (2) do influence microbial succession, the present study would imply that AHLs and additional bacterial factors are involved. We will investigate the mechanisms behind this effect in future studies.



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  		FIG. 3. DGGE showing the diversity of organisms that colonized abiotic control tubing containing lake water or LB broth or unfilled tubing. DGGE patterns of DNA from adherent bacteria (a) and a mixture of isolates (i) grown on R2A agar are also shown.


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ACKNOWLEDGMENTS
 
This research was sponsored by the U.S. Environmental Protection Agency (EPA) under grant application GR825503-01-0 and NIH grant R15 AI050638 (R.J.C.M.). The National Science Foundation and the United States Department of Agriculture provided support for C.F. (MCB-0223724 and CRI 2002-35319-12636).

We thank Christa Bates, Glenn Longley, Sue Reilly, Allana Welsh, and Ben Zhan for assistance and Grant Balzer and Andrew Hawkins for helpful comments.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Texas State University—San Marcos, 601 University Drive, San Marcos, TX 78666. Phone: (512) 245-3365. Fax: (512) 245-8713. E-mail: McLean{at}txstate.edu. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org. Back

{ddagger} Present address: QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 19355. Back


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Applied and Environmental Microbiology, December 2005, p. 8987-8990, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8987-8990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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