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Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.
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Bacterial life includes stages where the cells are associated and form a biofilm on a surface (e.g. Costerton et al., 1995). The formation of these surface communities and their inherent resistance to antimicrobial agents are the cause of many persistent and chronic infections (Costerton et al., 1999). Microscopic analysis has indicated that biofilm formation occurs in a sequential process of (i) transport of microbes to a surface; (ii) initial attachment; (iii) formation of microcolonies; and (iv) biofilm maturation (e.g. Tolker-Nielsen et al., 2000; Sauer et al., 2002). The biofilm formed by Pseudomonas aeruginosa under flowthrough conditions was found to be heterogeneous with mushroom-shaped microcolonies in studies in which glucose was used as the carbon source (e.g. Stewart et al., 1993; Davies et al., 1998), and flat, uniform and densely packed in studies where citrate was used as the carbon source (e.g. Heydorn et al., 2000; 2002), suggesting that P. aeruginosa biofilm development is dependent on the carbon source.
The use of a simple high-throughput screening assay for biofilm development has greatly facilitated the analysis of the genetic elements involved in biofilm formation. In this assay, bacteria grow under static conditions in the wells of microtitre plates and may form a biofilm on the abiotic well surface if they possess the necessary genetic elements. Quantification of the amount of biofilm formed in each well is done after removal of planktonic cells and appropriate staining of the surface-attached cells. Over the past few years, this assay has been used extensively to identify genes involved in the initial phases of biofilm formation in a number of bacteria, including the opportunistic pathogens Staphylococcus epidermidis (Heilmann et al., 1996) and Pseudomonas aeruginosa (O’Toole and Kolter, 1998a), which have become the model organisms for Gram-positive biofilm and Proteobacterial biofilm.
The use of flow chamber technology, fluorescent reporter genes and confocal laser scanning microscopy (CLSM) has enabled very detailed studies of specific and general interactions between the members of biofilm communities (e.g. Møller et al., 1998; Nielsen et al., 2000), and has permitted non-destructive studies of the dynamics and developmental steps occurring during biofilm formation (e.g. Tolker-Nielsen et al., 2000). In this set-up, the bacteria grow under hydrodynamic conditions and form biofilms on a glass surface. Over the past few years, this set-up has been used to study biofilm formation by a number of bacteria, including P. aeruginosa (e.g. Heydorn et al., 2002; Whitchurch et al., 2002).
Pseudomonas aeruginosa is able to swim in liquid by means of flagella and to move on surfaces by means of type IV pili (Semmler et al., 1999). The surface-associated so-called twitching motility is powered by extension and retraction of type IV pili (Skerker and Berg, 2001). Using the microtitre plate assay, O’Toole and Kolter (1998a) showed that flagella or flagellum-driven motility is required for biofilm formation by P. aeruginosa PA14, and that type IV pili are required for biofilm and microcolony formation by this organism. It was speculated that swimming motility might enable the bacteria to overcome repulsive forces at the surface–water interface so that they reach the surface, and that the microcolonies may be formed by twitching motility-driven cell aggregation. Using the flow chamber set-up, Heydorn et al. (2002) showed that a twitching motility-deficient mutant derivative of P. aeruginosa PAO1 is capable of both biofilm and microcolony formation. In fact, under the conditions used by Heydorn et al. (2002), the wild-type P. aeruginosa PAO1 formed a uniform flat biofilm without microcolonies, whereas the twitching motility mutant formed a biofilm with numerous microcolonies.
These apparently contrasting observations suggested that biofilm studies are sensitive to strain and experimental differences. The effect of experimental conditions on the factors required for biofilm formation was shown directly for Pseudomonas fluorescens. Non-motile P. fluorescens mutants were deficient in biofilm formation in the wells of microtitre plates when grown in minimal medium with glucose and casamino acids but, when the same mutants were grown in minimal medium with citrate, they did form biofilms in the wells of the microtitre plates (O’Toole and Kolter, 1998b). Consequently, there may be many pathways involved in biofilm development in these organisms. The different medium conditions that promote biofilm formation, and the different subsets of genes required under each condition, may indicate that various environmental niches are colonized by the bacteria through biofilm formation via different pathways.
Despite the evidence that P. aeruginosa biofilm development may be dependent on the carbon source used to support growth, and the finding that type IV pili are not always necessary for P. aeruginosa biofilm or microcolony formation, there is currently only one generally accepted model for P. aeruginosa biofilm development. According to this model, flagella mediate transport of the bacteria to the surface, type IV pili-driven motility along the surface leads to cellular aggregation and microcolony formation, and the subsequent formation of larger sessile mushroom-shaped multicellular structures occurs via a maturation process that requires cell-to-cell signalling (e.g. Costerton et al., 1999; O’Toole et al., 2000; Stoodley et al., 2002). In the present study, we show that, dependent on the type of carbon source, P. aeruginosa may form a biofilm with mushroom-shaped structures or a flat biofilm, and we carry out a characterization of the formation of the flat P. aeruginosa biofilm, which leads to an alternative biofilm model.
For the analysis with laser scanning microscopy, the bacteria needed to be fluorescently tagged. Fluorescent tagging of the strains was done with insertion of gfp, cfp or yfp at a defined genetically neutral chromosomal locus using mini-Tn7 constructs.
Initially, we tested the effect of the carbon source on the biofilm structure formed by P. aeruginosa PAO1 in our flow chamber set-up. The biofilms formed by Gfp-tagged P. aeruginosa PAO1 in flow chambers irrigated with minimal glucose medium were heterogeneous with mushroom-shaped microcolonies (Fig. 1A), whereas the biofilms formed in flow chambers irrigated with minimal citrate medium, minimal casamino acids medium or minimal benzoate medium were flat and densely packed (Fig. 1B–D). comstat image analysis on 18 CLSM images acquired at random locations in two glucose-grown, two casamino acids-grown, two citrate-grown and two benzoate-grown P. aeruginosa biofilms showed that the structural differences were highly significant (data not shown). Although the P. aeruginosa biofilm with mushroom-shaped multicellular structures has become widely accepted as the universal biofilm model (e.g. Costerton et al., 1999; O’Toole et al., 2000; Stoodley et al., 2002), there is clearly also a need to understand the biology of the flat biofilm that P. aeruginosa forms under some conditions. For example, it is not known what type of P. aeruginosa biofilm causes various persistent infections. The present work is an investigation of the development of the flat P. aeruginosa biofilm in flow chambers irrigated with citrate minimal medium. A similar analysis of the development of the heterogeneous P. aeruginosa biofilm in flow chambers irrigated with glucose minimal medium is ongoing in our laboratory.
Characterization of P. aeruginosa wild-type biofilm development and dynamics
In order to investigate P. aeruginosa biofilm formation and dynamics, we inoculated a flow chamber with a 1:1 mixture of Cfp-tagged and Yfp-tagged PAO1 wild-type cells, irrigated the flow chamber with citrate minimal medium and followed the biofilm development with time-lapse CLSM. As shown in Fig. 2A–D, microcolonies consisting of either Cfp-tagged or Yfp-tagged cells formed initially by clonal growth. After a period of initial microcolony formation by cell proliferation at fixed locations, the bacteria spread out on the substratum (Fig. 2E) and, subsequently, a flat biofilm covering the entire substratum was formed (Fig. 2F).
Bacterial migration along a surface in a biofilm may be type IV pili driven (O’Toole and Kolter, 1998a) or flagellum driven (Pratt and Kolter, 1998; Watnick and Kolter, 1999). In order to study the role of type IV pili and flagella in the formation of the flat P. aeruginosa biofilm, we derived the mutants ΔpilA, ΔfliM and ΔpilAΔfliM from P. aeruginosa PAO1 by allelic displacement.
Comparative analysis of adhesion of P. aeruginosa wild type and motility mutants
The ability of the wild type, ΔpilA, ΔfliM and ΔpilAΔfliM mutants to adhere to the glass surface in flow chambers with citrate minimal medium was tested under flowthrough conditions and under static conditions. As shown in Fig. 3A, the wild type and motility mutants adhered equally well in flow chambers under hydrodynamic or static conditions, indicating that flagella and type IV pili do not play a role in attachment of P. aeruginosa to a glass surface in flow chambers with minimal citrate medium. As it was shown previously that flagella and type IV pili mutants of P. aeruginosa PA14 were deficient in biofilm formation under static conditions in the wells of microtitre plates with glucose–casamino acids minimal medium (O’Toole and Kolter, 1998a), we tested the ability of our P. aeruginosa PAO1 wild-type, ΔfliM, ΔpilA and ΔpilAΔfliM strains to form biofilm in the wells of microtitre plates with glucose–casamino acids minimal medium and found that the ΔfliM, ΔpilA and ΔpilAΔfliM mutants were deficient in biofilm formation in comparison with the P. aeruginosa PAO1 wild type (Fig. 3B). There is therefore no reason to assume that P. aeruginosa PAO1 and PA14 use different pathways for biofilm formation. In microtitre plates with citrate minimal medium, however, the P. aeruginosa PAO1 wild type, ΔfliM, ΔpilA and ΔpilAΔfliM mutants all formed biofilms equally well (Fig. 3B), again suggesting that flagella and type IV pili do not play a role in surface attachment of P. aeruginosa in citrate minimal medium. The increased biomass in the glucose–casamino acids-grown wild-type biofilm in comparison with the citrate-grown biofilms (Fig. 3B) correlated with a higher yield in the glucose–casamino acids medium (data not shown).
Comparative analysis of biofilm architectures formed by P. aeruginosa wild type and motility mutants
The wild type and motility mutants were grown as biofilms in flow chambers irrigated with citrate minimal medium, and development was investigated by acquiring CLSM micrographs at days 1, 4 and 7 and subjecting the digital three-dimensional images to comstat image analysis. As shown on the representative CLSM micrographs in Fig. 4, and from the quantitative data in Fig. 5, the wild type and motility mutants formed biofilms with very different structures. The wild type formed a flat carpet, the ΔfliM mutant formed a hilly biofilm, and the ΔpilA and ΔpilAΔfliM mutants formed biofilms with irregular and protruding structures. Figure 5 shows roughness and average thickness quantification data for the four strains at days 1, 4 and 7. Each spot represents values calculated from a single CLSM image stack. The graphs therefore give both an impression of development over time and the variation within biofilm quantification data. The variation within strain is highest for the double mutant. A small variation within groups is dependent on structures small enough to be repeated within the microscope field. This can explain the fairly high variation between images of the large ΔpilAΔfliM mutant biofilm structures.
Comparative analysis of development and dynamics in biofilms of P. aeruginosa wild type and motility mutants
The differences in biofilm structure found for the wild-type, ΔpilA, ΔfliM and ΔpilAΔfliM strains suggested that bacterial motility plays a role in biofilm development. Presumably, the combination of growth and non-motility created the protruding structures in the mutant biofilms, whereas the combination of growth and migration resulted in the flat wild-type biofilm. In order to investigate this proposed role of motility in P. aeruginosa biofilm development further, we performed time-lapse CLSM in two-coloured wild-type, ΔpilA and ΔfliM biofilms, which had all been initiated with a 1:1 mixture of Cfp-tagged and Yfp-tagged cells. Microcolonies consisting of either Cfp-tagged or Yfp-tagged cells initially formed in all three biofilms (Fig. 6A–C). A small number of cyan fluorescent cells could sometimes be observed in yellow fluorescent microcolonies and vice versa, but extensively mixed microcolonies were not observed. After the initial microcolony formation, the wild-type and ΔfliM strains spread on the substratum, whereas the cyan or yellow fluorescent ΔpilA microcolonies grew bigger at fixed locations, suggesting that expansion on the substratum by the wild-type and ΔfliM strains was type IV pili driven. In the 4-day-old wild-type and ΔfliM biofilms, regions with Cfp-tagged and Yfp-tagged cells totally mixed occurred often (Fig. 6D and E), indicating that extensive motility occurred during biofilm formation. The 4-day-old ΔpilA biofilm did not contain mixed areas (Fig. 6F), supporting further the proposal that the majority of the motility occurring during wild-type and ΔfliM biofilm development was type IV pili driven.
Twitching motility as a spreading mechanism was also suggested by quantitative analysis of substratum coverage in wild-type, ΔfliM, ΔpilA and ΔpilAΔfliM biofilms. As shown in Fig. 7A, the wild type and ΔfliM mutants quickly covered the entire substratum, whereas the maximum substratum coverage reached by the ΔpilA and ΔpilAΔfliM mutants was about 50%.
Competition and selection in P. aeruginosa biofilms
The very dynamic nature of the P. aeruginosa biofilms investigated here suggested that competition and selection could occur extensively in the biofilms. Quantification of biomass accumulation showed that the ΔfliM and ΔpilAΔfliM biofilms accumulated more biomass than the wild-type and ΔpilA biofilms (Fig. 7B). As this indicated that the ΔfliM mutant could have a selective advantage in a dynamic biofilm, we investigated competition and selection in biofilms that had been initiated with a 1:1 mixture of Yfp-tagged wild type and Cfp-tagged ΔfliM mutant. Six CLSM images were acquired at six randomly chosen positions every 30 min for the first 24 h of biofilm growth, and biomass accumulation of the yellow fluorescent wild type and the cyan fluorescent ΔfliM mutant was quantified by comstat analysis. As shown in Fig. 8, immediately after inoculation, the number of wild-type cells and ΔfliM cells was roughly equal and, thereafter, the ΔfliM mutant rapidly outcompeted the wild type. Analysis of the absolute cell numbers indicated that a substantial fraction of the wild-type cells left the substratum during the first 2 h of biofilm growth, and that the wild-type cells then grew at the substratum at the same rate as the ΔfliM mutant for about 10 h, after which a fraction of the produced wild-type cells continuously left the biofilm in the rest of the analysed time period (data not shown). The opposite colour combination was also tested and gave the same results (data not shown).
Pseudomonas aeruginosa has become the model organism for proteobacterial biofilms, and much work has been done in order to identify consensus in biofilm formation and to formulate a general biofilm model (for recent reviews, see Costerton et al., 1999; O’Toole et al., 2000; Stoodley et al., 2002). The present study shows that P. aeruginosa biofilm formation is dependent on the carbon source used to support growth. A flat P. aeruginosa biofilm was formed when citrate, benzoate or casamino acids was used as carbon source, and a heterogeneous P. aeruginosa biofilm with mushroom-shaped multicellular structures was formed when glucose was used as the carbon source. The generally accepted model for P. aeruginosa biofilm development is based largely on studies of the heterogeneous P. aeruginosa biofilm (e.g. Costerton et al., 1999; O’Toole et al., 2000; Stoodley et al., 2002) and does not explain the formation of the flat P. aeruginosa biofilm. It seemed therefore that characterization of the developmental steps leading to the formation of the flat P. aeruginosa biofilm was needed, and therefore the present work was carried out. Our results lead to the alternative biofilm development model, which is shown as a schematic in Fig. 9 and is discussed below.
Our studies with P. aeruginosa PAO1 wild-type, ΔfliM, ΔpilA and ΔpilAΔfliM strains suggested that flagella and type IV pili are not necessary in the adhesion process in flow chambers or microtitre plate wells with citrate minimal medium. In microtitre plate wells with glucose–casamino acids medium, however, the ΔfliM and ΔpilA mutants were deficient in biofilm formation in agreement with the findings of O’Toole and Kolter (1998a) with P. aeruginosa PA14 derivatives. Defects in biofilm formation in glucose–casamino acids medium, but unhindered biofilm formation in citrate minimal medium, was also reported for motility mutants of P. fluorescens (O’Toole and Kolter, 1998b). Heydorn et al. (2002) found that a P. aeruginosa ΔpilHIJK mutant adhered five times better than the wild type in flow chambers with citrate. The pilHIJK genes, however, encode chemotaxis-related genes (Darzins, 1994) and, although a ΔpilHIJK mutant is twitching motility deficient (Darzins and Russell, 1997; Heydorn et al., 2002), it has not been shown that this mutant is non-piliated. In a study by DeKievit et al. (2001), adhesion of P. aeruginosa PAO1 was reported to be dependent on flagella and type IV pili under static conditions, whereas type IV pili and decreased flagellar motility did not affect biofilm formation of P. aeruginosa PAO1 in flow chambers with citrate medium or glucose medium. Another study showed that a non-flagellated mutant of the P. aeruginosa PA14 strain attached poorly in comparison with the wild type in a flowthrough system with glutamic acid medium (Sauer et al., 2002). A critical factor for transport of bacteria to the substratum in flowthrough systems is presumably the degree of turbulence near the substratum and, as the flow in different flowthrough systems may be more or less laminar/turbulent near the substratum, comparisons of bacterial adhesion in different flowthrough systems may be problematic.
Initial microcolony formation
Based on genetic and microscopic analysis, it has been proposed that the initial microcolonies in P. aeruginosa biofilms form by aggregation of bacteria via twitching motility (O’Toole and Kolter, 1998a), and that the initial microcolonies in Vibrio cholerae El Tor biofilms form by aggregation of cells via flagella-driven motility along the substratum (Watnick and Kolter, 1999). If a biofilm is initiated with a 1:1 mixture of yellow and cyan fluorescent bacteria, the formation of microcolonies through aggregation of the bacteria would result in mixed microcolonies containing roughly equal numbers of yellow and cyan fluorescent bacteria. Here, we report that biofilms that were initiated with a 1:1 mixture of yellow and cyan fluorescent bacteria, after an initial growth period, consisted of small microcolonies with predominantly yellow or predominantly cyan fluorescent cells. This suggests that the initial microcolonies were formed by clonal growth from single cells attached to the substratum, and that the formation of initial microcolonies through aggregation of bacteria does not play a significant role in P. aeruginosa biofilms under the conditions used in the present study.
After the initial formation of microcolonies by proliferation of sessile cells, the bacteria spread out on the substratum. CLSM time-lapse movies of development in the wild-type and ΔfliM biofilms indicated that the shift from sessile to motile cells occurred when the initial microcolonies reached a certain size, suggesting that the shift was induced by some sort of nutrient limitation. The fact that the bacteria in the ΔpilA and ΔpilAΔfliM biofilms did not spread on the substratum but instead formed protruding microcolonies at fixed locations suggested that the expansive migration in the wild-type and ΔfliM biofilms was type IV pili driven. Evidence has been presented that twitching motility may be stimulated by iron limitation (Singh et al., 2002), and that it may possibly be directed by chemical gradients (Darzins, 1994; Kearns and Shimkets, 1998; Kearns et al., 2001). However, although it is known that twitching motility in P. aeruginosa is regulated by Vfr (a homologue of the Escherichia coli cyclic AMP receptor protein, Crp; Beatson et al., 2002), by the RpoN-dependent two-component sensor regulator pair PilS/PilR (Hobbs et al., 1993; Strom and Lory, 1993) and by the FimS/AlgR sensor regulator pair (Whitchurch et al., 1996), the environmental cues that affect twitching motility in P. aeruginosa are still not well understood (Mattick, 2002). As twitching motility is powered by a mechanism involving extension–attachment–retraction of type IV pili (Skerker and Berg, 2001), it is possible that type IV pili also play a role in keeping the migrating bacteria surface associated. Yet, other cell-to-substratum and cell-to-cell connections keep the bacteria associated in the ΔpilA and ΔpilAΔfliM biofilms. The finding that the initial microcolonies in the ΔfliM biofilms did not flatten completely during biofilm development also suggested that flagellum-driven motility played a role in the formation of the flat wild-type biofilm (see below).
Biofilm maturation and dynamics
We followed biofilm development by the P. aeruginosa wild type and motility mutants for 7 days. Although the accumulation of biomass in the biofilms had not reached a plateau at day 7, the basic structures of the biofilms (flat, hilly, heterogeneous protruding) did not change in the following days and, therefore, we refer to the 7-day-old biofilms as the mature biofilms. The different structures of the wild-type, ΔfliM, ΔpilA and ΔpilAΔfliM mature biofilms suggested that flagella and type IV pili have roles in shaping the architecture of P. aeruginosa biofilms, although they are not necessary for biofilm formation. In the wild-type and ΔfliM mature biofilms, which were both initiated with a 1:1 mixture of Cfp-tagged and Yfp-tagged cells, the foci at which the initial microcolonies had formed still contained predominantly cyan fluorescent or yellow fluorescent cells, whereas the regions between these foci contained a mixture of cyan fluorescent and yellow fluorescent cells. Time-lapse CLSM suggested that production of new cells in the biofilms occurred at a high rate in the foci where the initial small microcolonies had formed. The formation of larger microcolonial structures in these foci in the wild-type biofilms was prevented as a result of type IV pili-driven bacterial migration, flagella-driven bacterial emigration out of the biofilm and probably also sloughing due to shear stress. The hilly structures in the ΔfliM mature biofilms were probably maintained in the mature biofilms because production of new cells in the foci at which the initial microcolonies had formed was not entirely counterbalanced as the ΔfliM bacteria were not able to carry out flagella-driven emigration out of the biofilm. Reduced emigration out of the ΔfliM biofilm is supported by the finding of higher biomass accumulation in the ΔfliM biofilms in comparison with the wild-type biofilms. The protruding elongated microcolonies in the ΔpilA and ΔpilAΔfliM mature biofilms most probably developed during growth because the cells were unable to spread on the substratum by means of twitching motility. CLSM time-lapse movies showing development of wild-type and ΔfliM biofilms, and illustrating the points discussed here, are accessible at the internet site http:www.im.dtu.dkmol-mic-avi.
In support of the role of twitching motility in biofilm development proposed here, Singh et al. (2002) showed that the presence of lactoferrin stimulates twitching motility through iron chelation and results in a flat P. aeruginosa biofilm, as opposed to a heterogeneous biofilm obtained under their conditions in the absence of lactoferrin. Although it was shown that the presence of lactoferrin did not inhibit growth, the flat biofilms found in the Singh et al. (2002) study were much thinner than the flat biofilms observed in the present study.
Gene expression in P. aeruginosa PAO1 biofilm cells and planktonic cells has been compared using DNA microarrays (Whiteley et al., 2001). The genes for synthesis of flagella and type IV pili were found to be repressed in the biofilm cells. The wild-type, ΔfliM, ΔpilA and ΔpilAΔfliM strains in the present study initially formed similar young biofilms with small microcolonies, but eventually formed very different mature biofilms, suggesting that flagella and type IV pili had roles in the later stages of biofilm development. Whiteley et al. (2001) used minimal casamino acids medium in their studies, which is shown here to give rise to a flat P. aeruginosa flow chamber biofilm similar to the citrate-grown flow chamber biofilm. Whiteley et al. (2001), however, used a chemostat vessel with granite pebbles as the substratum for biofilm formation, and it cannot be excluded that P. aeruginosa biofilm development in such a set-up differs from P. aeruginosa biofilm development in flow chambers.
Competition and selection in biofilms
The finding that the P. aeruginosa wild-type and ΔfliM biofilms are dynamic compositions with extensive migration occurring during development suggested that mutants that have a selective advantage in biofilms may outcompete less fitted variants. Competition experiments, in which biofilms were initiated with 1:1 mixtures of Yfp-tagged ΔfliM mutant and Cfp-tagged wild type (and the opposite colour combination), and the biomass accumulation of each strain in the developing biofilm was quantified by comstat analysis, showed that the ΔfliM mutant ousts the wild type. This suggests that biofilm experiments, especially those performed in flowthrough systems, should also be regarded as selection experiments. That is, the physiological state of a population in a mature biofilm could be the result of a selection process instead of a differentiation process as assumed by most authors (e.g. O’Toole et al., 2000; Stoodley et al., 2002). P. aeruginosa isolates from the lungs of cystic fibrosis patients are often non-flagellated (Luzar et al., 1985; Mahenthiralingam et al., 1994), indicating that selection for non-flagellated variants may occur in the biofilms in the lungs of cystic fibrosis sufferers.
A model for P. aeruginosa biofilm formation, which suggests the involvement of flagella in attachment, the requirement of type IV pili-driven motility in microcolony formation and the subsequent formation of larger microcolonial structures via a maturation process, has become widely accepted as a general model for P. aeruginosa biofilm development (e.g. Costerton et al., 1999; O’Toole et al., 2000; Stoodley et al., 2002). The present investigation of citrate-grown P. aeruginosa flow chamber biofilms suggests an alternative model for biofilm development shown schematically in Fig. 9. In this model, flagella play no role in attachment, initial microcolony formation occurs by clonal growth, and twitching motility thereafter causes spreading over the substratum and prevents the formation of larger microcolonial structures in the very dynamic flat mature biofilm. It appears that biofilm development occurs differently under different nutritional and environmental conditions, and that biofilm models with present knowledge can only be contextual.
Bacterial strains and media
The strains used in the study are listed in Table 1. Escherichia coli MV1190 was used for cloning, while E. coli HB101 and CC118(λpir) were used in matings. Modified FAB medium (Heydorn et al., 2000) was used supplemented with 10 mM citrate for batch overnight cultures, and with 0.1 mM citrate, 0.3 mM glucose, 0.015% casamino acids or 0.5 mM benzoate for biofilm cultivation. Biofilms and batch cultures were grown at 30°C.
Table 1. . Strains and plasmids used in the study.
Gmr Aprmob+; delivery plasmid for mini-Tn7-Gmr-PA1/04/03-ecfp
Gmr Aprmob+; delivery plasmid for mini-Tn7-Gmr-PA1/04/03-eyfp
Recombinant DNA techniques
The preparation of chromosomal and plasmid DNA, restriction endonuclease digestion, ligation reactions, polymerase chain reaction (PCR) and Southern blotting were carried out using standard protocols (Ausubel et al., 1992).
Construction of plasmids used for allelic displacement
The pilA::Telr cassette was constructed by: (i) PCR amplifying the pilA gene from genomic DNA of P. aeruginosa PAO1 using the primer pair 5′-ggaatcgaattcagaagtacgcggtcacctg-3′/5′-cggagatgcctacaaagagc-3′; (ii) cloning the blunted and EcoRI-digested 1.6 kb PCR fragment, into EcoRI–SmaI-digested pUC18-NotI; and (iii) replacing a 71 bp KpnI–BstXI fragment from the coding region of the pilA gene with a tellurite resistance gene obtained from pUT-Tel (Sanchez-Romero et al., 1998) as a blunted BamHI–HindIII fragment. The pilA knock-out plasmid, pTTN80, was constructed by cloning the pilA::Telr-containing NotI fragment in the NotI site of pCK318. The fliM::Tcr cassette was constructed by: (i) PCR amplifying two separate parts of the fliM gene from genomic DNA of P. aeruginosa PAO1 using the primer pair 5′-ggaatcaagcttcggcctgytgctggcgatcg-3′/5′-ggaatcggatccattt ccagggtcggcatgcg-3′ for the left 607 bp fragment and the primer pair 5′-ggaatcgagctccgctgttcatcctcgacgcc-3′/5′-ggaat cgaattcagsgccaggttgcccttgtg-3′ for the right 594 bp fragment; and (ii) cloning the PCR fragments, digested with BamHI and HindIII or SacI and EcoRI, in pTTN60 digested sequentially with the same restriction enzymes on either site of the Tcr gene. The fliM knock-out plasmid, pTTN61, was constructed by cloning the fliM::Tcr-containing NotI fragment in the NotI site of pCK318.
Construction of ΔfliM, ΔpilA and ΔfliMΔpilA mutant derivatives of P. aeruginosa PAO1 by allelic displacement
The P. aeruginosa ΔfliM, ΔpilA and ΔfliMΔpilA mutants were constructed by allelic displacement using triparental mating as described previously (Andersen et al., 1998) with E. coli HB101/RK600, P. aeruginosa PAO1 and E. coli CC118(λpir) containing delivery vector pTTN61 or pTTN80. Exconjugants with knock-out constructs inserted in the chromosome were selected on AB plates supplemented with citrate (10 mM) and tellurite (150 µg ml−1) or tetracycline (80 µg ml−1). Subsequent sucrose-based screening for double cross-over mutants was performed as described by Schweizer and Hoang (1995). Allelic displacement was confirmed by Southern analysis and PCR. As opposed to the wild type, the ΔfliM and ΔfliMΔpilA mutants did not spread in LB medium solidified with 0.3% agar, and did not show swimming motility under microscopic inspection. The presence of flagella on the wild type and the absence of flagella on the ΔfliM and ΔfliMΔpilA mutants were confirmed using Leifson's flagella staining method (Meynell and Meynell, 1970). As opposed to the wild type, the ΔpilA and ΔfliMΔpilA mutants did not spread by twitching motility on the interstitial surface between 1.5% agar and the Petri dish plastic surface. P. aeruginosa mutants inactivated in the major pilin subunit gene, pilA, are reported to be non-piliated (Mattick, 2002).
Construction of plasmids used for fluorescent tagging
The delivery plasmids miniTn7(Gm)PA1/04/03-ecfp-a and miniTn7(Gm)PA1/04/03-eyfp-a were constructed by: (i) PCR amplifying the ecfp and eyfp genes as 740 bp fragments from the templates pECFP and pEYFP (Clontech) using the primer pair 5′-atatagcatgctgagcaagggcgaggagctg-3′/5′-ctctc aagcttattacttgtacagctcgtccatgcc-3′; (ii) cloning SphI–HindIII-digested PCR fragments into the SphI–HindIII site of pTTN50; and (iii) cloning the 2000 bp NotI fragments containing the PA1/04/03-ecfp and PA1/04/03-eyfp fusions into the NotI site of pBK-miniTn7-ΩGm.
Insertion of cfp, gfp and yfp into the chromosome of P. aeruginosa PAO1,ΔfliM,ΔpilA andΔfliMΔpilA
In order to insert ecfp, egfp or eyfp into a neutral intergenic region downstream of the glmS gene in the P. aeruginosa genome, we used the mini-Tn7 system described by Koch et al. (2001). Four-parental mating was carried out with the relevant P. aeruginosa strain, E. coli HB101/RK600, E. coli HB101/pUX-BF13 and E. coli HB101 containing pBK-mini-Tn7-gfp2, miniTn7(Gm)PA1/04/03-ecfp-a or miniTn7(Gm)PA1/04/03-eyfp-a. The egfp, ecfp and eyfp genes are fused to the PA1/04/03 promoter (Lanzer and Bujard, 1988), which acts as a constitutive promoter in Pseudomonas spp. (Andersen et al., 1998). Fluorescent exconjugants with mini-Tn7 cassettes inserted in the chromosome were selected on AB plates supplemented with citrate (10 mM) and gentamicin (30 µg ml−1). The correct insertion of the mini-Tn7 cassettes in the intergenic region immediately downstream of the glmS gene was confirmed by Southern analysis and PCR. The fluorescently tagged strains showed no phenotypic changes compared with the parental strains, when tested in liquid medium or flow chamber biofilms.
Cultivation of biofilms
Biofilms were grown at 30°C in flow chambers with individual channel dimensions of 1 × 4 × 40 mm. The flow system was assembled and prepared as described previously (Møller et al., 1998). The flow chambers were inoculated by injecting 350 µl of overnight culture diluted to an OD600 of 0.001 into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow was started using a Watson Marlow 205S peristaltic pump. Mean flow velocity in the flow cells was 0.2 mm s−1, corresponding to laminar flow with a Reynolds number of 0.02.
To quantify attachment under flowthrough conditions, 350 µl of overnight cultures (wild type, ΔfliM, ΔpilA and ΔfliMΔpilA) diluted to OD600 = 0.1 was injected into two channels with the medium flow running (0.2 mm s−1). After 5 min with medium flow running, nine CLSM images were captured at random positions in each of the flow channels. To quantify attachment under static conditions, 350 µl of overnight cultures (wild type, ΔfliM, ΔpilA and ΔfliMΔpilA) diluted to OD600 = 0.001 was injected into flow chambers without flow. After 1 h, the flow was started (0.2 mm s−1), and nine CLSM images were captured at random positions in each flow channel. The number of adherent bacteria was subsequently counted by subjecting the CLSM images to image analysis.
Microtitre plate biofilm formation assay
The microtitre plate biofilm formation assay was performed according to the method of O’Toole and Kolter (1998a) with modifications. Overnight cultures were grown in modified FAB medium supplemented with 0.2% glucose + 0.5% casamino acids, or 10 mM citrate, and diluted to OD600 = 0.001 with fresh medium. The diluted cultures (100 µl) were transferred to wells of polystyrene microtitre plates and incubated for 48 h at 30°C. Biofilm cells were stained using a Biomek 2000 laboratory automation workstation from Beckman Coulter, which (i) washed the wells twice with 0.9% NaCl; (ii) stained with 0.1% crystal violet; (iii) washed twice with 0.9% NaCl; and (iv) resuspended in 96% ethanol. Biofilm cell-associated dye was measured at OD600.
Microscopy and image acquisition
All microscopic observations and image acquisitions were performed on a Zeiss LSM 510 confocal laser scanning microscope equipped with detectors and filter sets for monitoring of Cfp, Gfp and Yfp fluorescence. Images were obtained with a 63 × 1.4 objective or a 40 × 1.3 objective. Simulated three-dimensional images, shadow projections and sections were generated using the imaris software package (Bitplane).
CLSM images were analysed by the computer program comstat (Heydorn et al., 2000). A fixed threshold value and connected volume filtration was used for all image stacks. A script written in matlab (MathWorks) was used to count attached cells from CLSM images.
This work was supported by the Danish Biotechnological Research Program. We thank Anne Nielsen for technical assistance with the construction of miniTn7(Gm)PA1/04/03-ecfp-a and miniTn7(Gm)PA1/04/03-eyfp-a.