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Chapter 13 : Temporal Segregation: Succession in Biofilms

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Abstract:

During the past 10 years or so there has been a rapidly increasing interest in bacterial biofilms. There are several reasons for this interest. First, active bacterial life outside the laboratory is predominantly associated with biofilms. Second, there are now excellent tools for detailed on-line studies of heterogeneous populations. Finally, the development of multicellular surface populations may share traits with developmental processes common in higher eukaryotic organisms. Biofilm development may be described as a stepwise multicellular differentiation process. Structured communities, such as biofilms, create heterogeneous conditions and allow subpopulation development in niches. The extraordinary capacity of bacteria to coordinate different parts of their biochemical repertoire is evident in biofilm development. The current ideas concerning the individual steps in biofilm development are discussed in detail. The major cause of biofilm physiological complexity is the development of diverse niches in the community. Phase variation creates physiologically different subpopulations in clonal populations. The direction of phase variation switches may be influenced by the external environment. Adaptive mutations induced by the environment seem to be particularly suited to the biofilm environment during its development and persistence. One of the major routes of gene transfer in the bacterial world is by DNA transformation, requiring cellular competence and some degree of DNA homology. Horizontal gene transfer may be one key determinant in the generation of substantial genetic alterations. The investigations of the adaptive and evolutionary potentials of bacteria in the context of complex communities in biofilms and other structured environments have only just started.

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13

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Figures

Image of FIGURE 1
FIGURE 1

Laboratory biofilm setups. (A) Microtiter plate with biofilms formed in the wells. Visualization of the biofilms is performed after removal of the culture liquid and staining of the attached biomass with crystal violet. (B) The flow-cell biofilm setup as used in the authors' laboratory. The medium containing nutrients for the biofilm is pumped from the reservoir vessel through bubble traps catching air bubbles to the inlet of the flow chamber. The medium runs through the flow-chamber channels (compare blowup), and biofilm is formed on a glass coverslip glued onto the flow chamber. Medium and nonattached cells are finally collected as an effluent in a waste flask. The flow chambers can be placed directly under the microscope for in situ observations.

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 2
FIGURE 2

Biofilm development. (A) A schematic model of biofilm formation on solid/liquid interfaces. The individual steps in the process are described in the text. The different shapes, greytones and shadings of the microcolonies symbolize the heterogeneity of cell physiology and EPS matrix composition. Planktonic swimming cells are indicated by the presence of polar flagella on the cells, twitching motility is symbolized by surface-attached cells with arrows, and, in all cases, the flow direction is from left to right as indicated in step 4. (B) Biofilm development monitored by scanning confocal laser microscopy (SCLM). Cells of were grown in a flow chamber (see Fig. 1 ), and the four frames show how the different steps indicated in part A develop over time. In panels a and b, the biofilm is viewed from above, showing the cells attached to the glass surface (a) and the beginning of cell proliferation (b). In panels c and d, the presentations are shadow projections from above showing further cell proliferation (c) and biofilm maturation (d). In the side frames of panels c and d, the biofilms arc viewed from the side to indicate the height of the microcolonies. This series of images only represents the first four steps of the model presented in part A. (These images were kindly provided by Janus Haagensen, BioCentrum-DTU.)

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 3
FIGURE 3

Spatial heterogeneity in biofilms. Section through microcolony in flow-chamber biofilm of with an inserted gene fusion between an promoter from and a gene encoding an unstable Gfp fluorescent protein. The two frames show the same microcolony with two different filter sets on the confocal microscope. (A) Staining with a ribosomal probe targeting rRNA in the cells. All cells light up with more-or-less the same intensity, indicating that the cells are distributed throughout the microcolony. (B) Gfp fluorescence expressed in the microcolony. Cells on the surface of the colony express strong green fluorescence, whereas those in the inner parts of the colony are nonfluorescent or show weak fluorescence. The use of an unstable Gfp reporter makes it possible to distinguish between glowing and nongrowing cells. (Reprinted with permission from .)

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 4
FIGURE 4

Two-species consortia growing as biofilms in flow chambers on a minimal buffered medium with benzyl alcohol as the only carbon and energy source. See text for details. (A) Schematic overview of benzyl alcohol degradation in the biofilm consortium shown in panel B. The arrow size in each step illustrates the degradation rate and the carbon flow between the two consortium members. (B) The two strains are sp. C6 (red cells) and R1 (blue or turquoise cells). Growth-active cells of R1 are turquoise (blue + green) due to the inserted activity reporter cassette containing a ribosomal RNA promoter in front of a gene encoding unstable Gfp protein (compare legend to Fig. 3 ). The side-frames are -sections of the biofilm in the positions indicated with white arrows. (C) As in panel A, but valid for the consortium shown in panel D. Note that KT2440 scarcely degrades benzyl alcohol and therefore is completely dependent on benzoate excreted from the strain. (D) Section of the biofilm containing the strains of the sp. C6 (red cells) and KT2440 (green cells), a derivative of mt-2. (E) The interactive coupling between sp. C6 and a mutant selected in the panel D consortium. In panel B, sp. C6 and are identified by fluorescence in situ rRNA hybridization using specific probes targeting the organisms. In panels C and D, the biofilm is stained with cyto62 (red), and additionally, was identified by Gfp expression.

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 5
FIGURE 5

Variants with altered cell surface or surface components often give rise to different colony morphologies. Three different colony types of are shown here. (A) The normal smooth wild-type colony growing on a broth agar surface. (B) A variant derived from biofilms; note the wrinkly appearance of the colony. (C) The colony is yet another morphotype, also isolated from a biofilm.

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 6
FIGURE 6

The life cycle of . This successful pathogen has evolved multiple strategies for adaptation and survival in changing environments. In aquatic environments the EPS producing phase variant (see text) may be a better survivor by forming biofilms on surfaces. When people drink water from sources colonized by there is a risk of infections of the gastrointestinal tract, this time selecting pathogenic variants expressing the cholera toxin. Infections with cause severe diarrhea, resulting in release of large numbers of bacteria back to the environment.

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 7
FIGURE 7

The occurrence of mutator strains in populations of . Clones of were collected from two groups of patients: (A) cystic fibrosis patients with chronic infections of and (B) patients not having cystic fibrosis with lung or blood infections. For each of the isolated clones the frequency of mutation to rifampicin resistance was determined by plating. It is clear that in the CF patients a significant subpopulation of bacteria are mutating with very high frequencies, whereas in the normal patients the determined mutation frequencies are in a range that is normally observed in growing bacteria (standard PAO1 strain included in frame A and indicated by an asterisk). The dashed lines represent the mean of mutator and nonmutator groups. (Reprinted with permission from .)

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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Image of FIGURE 8
FIGURE 8

In situ monitoring of plasmid transfer. (A) Design of a mating-pair system. The donor strain harbors a conjugative plasmid with an inserted fusion between a promoter and a promoterless gene. This strain also has a chromosomal insert of the gene expressing a repressor, which prevents expression of Gfp from the plasmid. The recipient strain has no repressor gene. After plasmid transfer the transconjugants will express Gfp (zygotic induction, no repression of the promoter), thus allowing identification of these as green fluorescent cells. (B) Plasmid transfer in a flow-chamber biofilm. In this demonstration of the mating-pair system described in A, the donor and recipient cells were both . The donor strain harbored the pWWO plasmid (TOL), which is highly efficient for transfer between pseudomonads. Transconjugant cells are green/yellow in the image, whereas no distinction between donors and recipients (all red) can be made in this case. (Reprinted with permission from , and .)

Citation: Hansen S, Molin S. 2004. Temporal Segregation: Succession in Biofilms, p 192-213. In Miller R, Day M (ed), Microbial Evolution. ASM Press, Washington, DC. doi: 10.1128/9781555817749.ch13
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