This raises the question as to whether LecB and CdrA are functionally interchangeable? Therefore, we examined if CdrA influenced Psl distribution and aggregate formation in our system. Expression of lecB in trans in the double mutant background was sufficient to restore wild-type aggregate formation and Psl distribution Fig. In contrast, expression of cdrA in the same background was not sufficient to restore wild-type biofilm characteristics. This strain produced a flat, thick mass devoid of aggregates Fig.
Taken together, these results suggest that when grown in NB, CdrA can influence biofilm structure and Psl retention, but LecB is the primary matrix protein responsible for Psl localization and aggregate production. Our previous work demonstrated a prominent role for CdrA in aggregate formation.
In attempting to reconcile our current results with this past study, we noted that the main culturing difference involved the growth medium. In this study, a dilute complex medium NB medium was used to mimic the previous work done on LecB On the other hand, our past work with CdrA involved dilute LB medium. Since our results suggest that LecB and CdrA can carry out similar roles in the matrix, we hypothesized that either CdrA or LecB expression levels may change significantly when P.
The idea being that LecB may not be required under conditions where CdrA is produced at a sufficiently high level or vice versa. To test this possibility we probed biofilm biomass harvested from the surface of silicone tubing using both CdrA and LecB antisera. LecB levels were observed to be roughly the same for the two growth media Supplementary Fig. These data indicate that LecB production is critical for aggregate formation under culturing conditions e.
The underlying cause of lower CdrA expression levels during growth on NB remains unclear and is a focus of future study. Our results indicate that LecB binds Psl and guides its localization within biofilm aggregates. Past work with CdrA demonstrated that its expression was linked to retention of cells and Psl to a growing biofilm. This led us to hypothesize that LecB might play a similar role during the course of biofilm growth. To test this hypothesis, we used the arabinose-inducible lecB expression strain and quantified the amount of cells and Psl released into the bulk liquid in the presence and absence of arabinose.
To facilitate quantitation of biofilm biomass, the biofilms were cultured in silicone tubes through which media was continuously flown. Initially, we verified by microscopy that the structure of the biofilm formed by the wild-type and lecB mutant strains were similar to their flow-cell counterparts Supplementary Fig.
LecB retains cells and matrix material in adherent biomass. Tubes are represented containing biomass green and media blue. Absence of LecB results in increased number of non-adherent cells compared with when LecB is present. Expression of lecB increases the number of cells adhered to the tube surface compared with when LecB is not present.
Immunodetection was performed using anti-Psl antibodies. Expression of lecB leads to decreased levels of released Psl when compared with the uninduced condition. Our analysis indicates that LecB expression had repercussions that extended beyond the ability to form aggregates.
We found that LecB had a profound impact on the ability to retain growing biomass and secreted Psl Fig. When LecB was expressed, the number cells was higher in the adherent fraction compared with when it was not expressed Fig. When assaying for Psl, we observed lower levels in the non-adherent fractions of the arabinose-induced strain, indicating that LecB also influences the retention of Psl Fig.
Taken together, these results show that LecB enhances the retention of both cells and Psl to the growing biofilm. Lectins are found in all domains of life, and their key function is often to mediate interactions 30 , In bacteria, lectins are nearly exclusively studied in the context of bacterium interactions with higher Eukaryotes 16 , 18 , 32 , 33 , Indeed, these interactions have critical roles in both disease and symbioses.
However, particularly in bacterial species, lectins functioning in the context of microbial communities has not been extensively explored. This is certainly the case for P. Our findings suggest that lectin-mediated interactions that stabilize the biofilm matrix represent a distinct function for at least one of these lectins. Biofilm formation usually involves the production of EPS and proteins that lend structural integrity to the matrix 5 , 35 , 36 , 37 , One of the better characterized systems demonstrating these principles involves Vibrio cholerae.
Three matrix proteins were identified in V. After the production of the main exopolysaccharide VPS 39 , a protein involved in cell—cell and cell—surface adhesion RmbA accumulates at the cell surface 40 , 41 , Next, Bap1 is secreted and is thought to crosslink unknown matrix components and cells to ensure matrix integrity, as well as to contribute to the hydrophobicity of the pellicle 41 , 42 , Last, RbmC accumulates at discrete sites and is crucial for retaining VPS throughout the biofilm 41 , 42 , These principles appear to be conserved in P.
Biofilm aggregates likely serve some key functions. For biofilms growing at a liquid—solid interface, aggregates protrude out of the boundary layer found at the surface and into the flow stream overlying bulk liquid.
One consequence of this is that cells positioned toward the top of the aggregate have favorable access to the overlying nutrients. Indeed, this point is described in a number of laboratory and computational studies of biofilms 45 , 46 , Aggregates also harbor the most antibiotic tolerant subpopulations of biofilm cells 48 , Thus, aggregates may represent the most protective structures present in a surface-associated community.
Therefore, the ability of biofilm communities to produce aggregates may be critical for obtaining the maximal fitness benefits of this growth state. When CdrA was first described, its function within the biofilm was to maintain the structural integrity of aggregates, in part, by promoting Psl localization to the aggregates periphery 3.
In this study, we show a very similar role for LecB. In addition, we know that a secreted, extracellular form of CdrA is capable of binding to Psl in the matrix and promoting matrix stability. The simplest interpretation may be that having functionally redundant or partially redundant matrix proteins ensures that deleterious mutations targeting either of their genes do not impair the production of aggregates.
Redundancy for critical functions is certainly a common theme encountered in P. However, our observations in NB medium indicate that it might not be that simple. Complementation with lecB restores wild-type biofilm formation, with large aggregates and Psl retained at the aggregate periphery of these aggregates. Curiously, complementation of the double mutant strain with cdrA failed to restore production of wild-type aggregates.
Psl was retained in the biofilm, but the biofilm was a thick homogenous mat of cells Fig. This result suggests that under some instances, simple expression of LecB or CdrA is not sufficient to support aggregate production.
Why is it unclear? Perhaps, the two proteins differ in their stability under changing environmental conditions. Finally, we cannot rule out that yet unidentified matrix proteins can also contribute to matrix stability in the absence of either LecB or CdrA. Another point of interest is that two clades of LecB have been proposed, one that groups with PAO1 and another that groups with PA14 Whether LecB can serve a similar role in Psl-binding and biofilm structure for other Psl-producing members of the PA14 clade remains to be determined.
We propose the following model to explain our experimental observations. Surface attachment is similar for both strains, resulting in the production of matrix components EPS and CdrA.
At this stage, cells proliferate and produce both CdrA at low levels and LecB which leads to the retention of Psl at the base of the biofilm. In the wild-type strain, aggregates continue to grow and biofilm biomass begins to extend beyond the boundary layer and into the overlying bulk fluid.
At this stage, lecB mutant strains are swept away by the shear stress resulting from fluid flow due to their inability to stabilize the aggregate through Psl interactions. While we are able to determine the consequences of the lack of LecB for biofilm maturation, our data do not explain why LecB and Psl begin to co-localize at the aggregates periphery. Our data provide a foundation for the current model, however, there are many questions still left unanswered. Future experimentation will include determining whether lecB and Psl expression are coordinately regulated.
However, our current knowledge suggests that Psl is primarily influenced by c-di-GMP signaling, while lecB is quorum sensing controlled. In conclusion, our study demonstrates that LecB can serve as a key structural protein in the biofilm matrix. We also demonstrated that Psl and LecB are binding partners and that this interaction impacts biofilm structure. Our findings also have implications for multi-species systems. We also predict that the converse may be true: P. Finally, we predict that lectin production within biofilms may allow P.
Growth curves were performed for all the backgrounds used and no growth defect was observed. All strains are listed in Supplementary Table 1.
Generation of mutants followed the previously published protocol All mutants were confirmed by sequencing using the primers UpF and DownR.
Insert was sequenced using the primer pBAD prom Fw. To purify LecB, P. The supernatant obtained after centrifugation was loaded onto a mannose agarose column Vector Laboratories. The precipitate was washed with ethanol, air-dried, resuspended in water, treated with DNaseI, RNase A, and proteinase K and subsequently lyophilized.
For the purification of Psl these additional steps were followed: first crude preparations were resuspended in water and exhaustively dialyzed using a MWCO Slide-A-Lyzer dialysis cassette. Then these polysaccharides were fractionated on a Sephadex G column and carbohydrate-containing fractions were screened by western immunoblotting. Procedures performed here were adapted from previously published protocol Plates were sealed and incubated in a damp box.
After coating, plates were washed five times. The coating efficiency of the different crude extracts was confirmed by titration with anti-Psl-specific antisera. Plates were washed five times. Binding was detected by incubation with anti-human IgG Fc-specific, horseradish peroxidase conjugate. Absorbance was measured using a FluorchemQ. Readings were measured against a blank of uncoated wells.
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Reynolds for technical assistance with electron microscopy, and S. Han and J. Zhang for mouse surgery. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper. We would like to thank all members of the Hauser, Ozer, and Kociolek laboratories for their valuable comments during numerous discussions of this work. Kelly E. Bachta, Jonathan P. Allen, Bettina H. Its metabolism is respiratory and never fermentative, but it will grow in the absence of O 2 if NO 3 is available as a respiratory electron acceptor.
The typical Pseudomonas bacterium in nature might be found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a unicellular organism, actively swimming by means of its flagellum. Pseudomonas is one of the most vigorous, fast-swimming bacteria seen in hay infusions and pond water samples. In its natural habitat Pseudomonas aeruginosa is not particularly distinctive as a pseudomonad, but it does have a combination of physiological traits that are noteworthy and may relate to its pathogenesis.
It is often observed "growing in distilled water", which is evidence of its minimal nutritional needs. In the laboratory, the simplest medium for growth of Pseudomonas aeruginosa consists of acetate as a source of carbon and ammonium sulfate as a source of nitrogen.
Organic growth factors are not required, and it can use more than seventy-five organic compounds for growth. It is resistant to high concentrations of salts and dyes, weak antiseptics, and many commonly used antibiotics. These natural properties of the bacterium undoubtedly contribute to its ecological success as an opportunistic pathogen. They also help explain the ubiquitous nature of the organism and its prominence as a nosocomial pathogen.
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