N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A. tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.
Autoinduction is a form of intercellular communication, in which cells monitor the level of self-produced autoinducer signal molecules, typically acylated homoserine lactone derivatives (AHLs). AHLs are membrane-permeant signal molecules that accumulate as a function of cell density, and at some threshold level, described as a bacterial quorum, activate expression of target genes via AHL-responsive transcriptional activators. Therefore, quorum-dependent gene expression occurs preferentially at high cell densities (reviewed in [1, 2]). Autoinduction was first described in Vibrio fischeri in which it controls bioluminescence through luxI encoding an AHL synthase, and an AHL-responsive transcriptional activator called luxR. luxR and luxI homologues have been described in a number of organisms including Agrobacterium tumefaciens in which it regulates conjugal transfer of the Ti plasmid [3, 4], and Pseudomonas aeruginosa in which it regulates extracellular virulence [5, 6]. While the basic structure of AHLs is conserved between different bacteria, they vary in the length and substitution of the acyl chain, thereby permitting specific regulation of some genes . Nevertheless, many genes can be activated by heterologous AHL autoinducers [7, 8] implying that bacteria can sense and react to population densities of their own species and population densities in general.
In their natural environments, bacteria frequently attach to surfaces. Growth and further colonization of a surface results in the development of an adherent microbial community referred to as a biofilm . Biofilms are very prevalent in nature having been described in a number of medical, industrial, and environmental contexts [9, 10]. Organisms within biofilms have been shown to be slow-growing because of cell density and limited access to nutrients , factors which may explain the high resistance of biofilm populations to antimicrobial agents . The high density of bacteria within biofilms has led to the speculation that quorum-sensing genes and AHL production may be fundamentally associated with biofilm physiology. At least in some bacteria, mutations in autoinducer synthesis lead to the formation of biofilms with abnormal structures (D. Davies, M. Parsek, J. Pearson, B.H. Iglewski, J.W. Costerton, and E.P. Greenberg, personal communication). Addition of AHL has been shown to accelerate the recovery of nitrifying biofilms from starvation . Here we document the presence of AHL activity in naturally occurring aquatic biofilms.
2Materials and methods
2.1Strains and growth conditions
A. tumefaciens A136 (Ti−) (pCF218)(pCF372)  was used as an indicator strain for exogenous AHL autoinducers. The sensitivity of this strain to AHL in a cross-feeding assay such as employed here has been estimated to be ≥3 nM 3-oxo(octanoyl) homoserine lactone, i.e. the Agrobacterium autoinducer (S.C. Winans, personal communication). A. tumefaciens KYC6 (traM::Tn5-gusA harboring pCF218) was used as an endogenous AHL overproducer . For long-term storage, all cultures were suspended in a mixture of LB Broth (Difco Laboratories, Detroit, MI) and glycerol and frozen at −80°C. Prior to use, frozen cultures were removed from storage, and incubated on AT medium  (strain KYC6) or AT medium supplemented with spectinomycin and tetracycline (strain A136) (pCF218)(pCF372) . All cultures were incubated at 30°C.
2.2Sample collection and processing
Small limestone rocks (1–2 cm diameter), coated with biofilms, were aseptically removed from the bed of the San Marcos River, a karst aquifer-fed river in south-central Texas, USA. Based on three previous studies (K.A. Dunn and R.J.C. McLean, unpublished) we estimated the concentration of bacteria on these rocks to be at least 108 CFU cm−2. Based on colony morphology on R2A agar (Difco), at least 30 different types of organisms were present, from which two, Pseudomonas putida and Pseudomonas fluorescens, were identified. Eukaryotic algae were also present within the biofilms, however they were not identified. Biofilm-coated rocks were processed within 30 min of sample collection. Limestone rocks (1–2 cm diameter) not associated with biofilms were collected from a local limestone outcropping. In order to preclude colonization of these control rocks by biofilms or endolithic microbial communities , these rocks were chipped from the interior (3 cm depth) of a dry limestone outcropping. The control rocks were used within 1 h of collection.
The experimental parameters used in this investigation are listed in Table 1. Biofilm extracts were obtained by placing rocks with naturally formed biofilms in sterile H2O, and sonicating them for 15 min in a bath sonicator. The cells were then removed by centrifugation at 17 000×g for 5 min. Similarly Agrobacterium AHL (3-oxo(octanoyl) homoserine lactone) was obtained from cell-free culture fluids of broth-grown A. tumefaciens KYC6. When rocks or biofilm sonicate were to be sterilized, they were autoclaved for 15 min at 121°C.
Table 1. Summary of experimental results
aA136 reporter strain was incubated with each substance as detailed in the text and demonstrated in Fig. 1.
bResults indicated by a blue coloration due to lacZ expression in A. tumefaciens A136. A + indicates an AHL concentration of ≥3 nM based on A. tumefaciens A136 sensitivity to 3-oxo(octanoyl) homoserine lactone, i.e. the Agrobacterium autoinducer (S.C. Winans, personal communication).
A136 (negative control)
KYC6 (positive control)
Autoclaved biofilm-coated rock (AB)
Autoclaved rock lacking biofilm (AL)
KYC6 culture supernatant
Autoclaved biofilm sonicate
Autoclaved KYC6 supernatant
2.3Cross-feeding assay for AHL detection
AT medium, covered with 50 ml X-gal (20 mg ml−1 stock solution in dimethyl formamide) was used for cross-feeding assays. These assays consisted of streaking the AHL reporter strain, A. tumefaciens A136 (pCF218)(pCF372), on the plate and then placing the rock or culture to be tested approximately 1 cm distant (Table 1, Fig. 1). If AHL activity was present, it would diffuse through the agar to the reporter strain and be detected due to activation of the traI-lacZ fusion by TraR . Positive and negative controls consisted of culturing the reporter strain with A. tumefaciens KYC6 (AHL overproducer) and A. tumefaciens A136 (reporter strain Ti− with a traR− deletion). A minimum of four replications were performed for each variable.
3Results and discussion
All experimental parameters and qualitative results for AHL activity are summarized in Table 1. When biofilm-covered rocks were incubated in the presence of the reporter strain, A. tumefaciens strain A136 (pCF218)(pCF372), it produced a blue coloration in the bioassay medium, due to active expression of the lacZ reporter gene. LacZ expression resulted from cross-feeding of the reporter strain with AHL synthesized by the biofilms, and activation of the traI-lacZ fusion by the TraR protein (Fig. 1A). This was indicative of AHL expression which was detected by the traI::lacZ fusion . Similar evidence of AHL activity was seen when A. tumefaciens A136 (pCF218)(pCF372) was cultivated with A. tumefaciens KYC6 (positive control) (Fig. 1B) or with either biofilm sonicate or KYC6 sonicate (Table 1). When biofilm-coated rocks or rocks which lacked biofilm were autoclaved prior to testing, no evidence of AHL activity was seen (Fig. 1C,D). No evidence of AHL activity was seen in the negative control (Fig. 1E). AHL activity could be restored if either KYC6 supernatant or biofilm supernatant was applied to these autoclaved rocks (Table 1). Autoclaving the KYC6 (pCF218) cell-free culture fluid did not abolish AHL activity. Therefore it is likely that the presence of AHL activity seen in biofilm-coated rocks (Fig. 1 A compared with Fig. 1C) is due to bacterial growth with concomitant AHL production during testing rather than detection of AHL which had diffused from the biofilm-coated rock. Detection of AHL in the cell-free biofilm sonicate indicates the presence of this preformed quorum sensor in the native biofilm.
While the distribution of biofilms is quite ubiquitous, many fundamental aspects of their physiology remain unknown. Studies with scanning confocal laser microscopy (SCLM) have shown biofilms to have a complex architecture and physiology . Bacteria within biofilms are often arranged in microcolonies, small clusters of bacteria typically containing 2–200 cells . Microcolonies are separated by water channels which are extracellular polysaccharide-containing regions containing relatively few cells. In mixed culture biofilms, microcolonies contain consortia of several species, which permits intercellular activities such as syntrophic metabolism of xenobiotics , genetic exchange [9, 17], and intracellular communication  to occur. SCLM studies show that many physiological functions in monoculture and mixed culture biofilms are performed by a small number of individual cells, a phenomenon that has led to the proposal that biofilm communities are analogous to tissues in higher organisms  and may represent an evolutionary step between unicellular non-specialized organisms and multicellular organisms which possess specialized cells. Similar descriptions of multicellular activity have been given for other bacterial systems, not associated with biofilms (reviewed in ). Several molecules have been associated with coordinate activity of microorganisms within a community. These include AHL, oligopeptides, amino acids such as glutamate and aspartate, and fatty methyl esters . It is conceivable that one or more of these molecular signals may impact biofilm physiology.
The reporter strain, A. tumefaciens A136 (pCF218)(pCF372), used in this study, has the ability to detect a wide variety of AHL molecules . It is unclear whether the AHL expression seen in this study is from all bacterial components within the biofilm or from a select few. It is also unclear whether several forms of AHL are being produced. Should several forms of AHL be produced, then this would permit intercellular communication within a biofilm which may be directed at the population as a whole or at individual populations within this community. We are currently pursuing further studies in this area.
We express our deep appreciation to E.P. Greenberg and J.W. Costerton for sharing some ideas and enthusiasm with us. This work was supported by the Biology Departments at Southwest Texas State University (R.J.C.M. and M.W.) and Trinity University (W.C.F.). R.J.C.M. dedicates this paper to James W. Lang.