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Novel imaging technologies for characterization of microbial extracellular polysaccharides.

Lilledahl MB, Stokke BT - Front Microbiol (2015)

Bottom Line: Understanding of biology is underpinned by the ability to observe structures at various length scales.This is so in a historical context and is also valid today.Evolution of novel insight often emerges from technological advancement.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology Trondheim, Norway.

ABSTRACT
Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects.

No MeSH data available.


Related in: MedlinePlus

High speed AFM imaging of Trocoderma reesei Cel7A cellulase acting on crystalline Iα cellulose. (A) AFM topographs of the same area of the cellulose crystal imaged at intervals of 0.9 s revealing different localization of individual cellulases. (B) Time dependence from the initial position of individual cellulase positions when acting on cellulose crystal as deduced by image processing of HS-AFM topographs. The colored symbols reflect data extracted from individual enzymes whereas the dotted line depict an average velocity as reported (Igarashi et al., 2009). Reprinted with permission from Igarashi et al. (2011). Copyright (2011) AAAS.
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Figure 3: High speed AFM imaging of Trocoderma reesei Cel7A cellulase acting on crystalline Iα cellulose. (A) AFM topographs of the same area of the cellulose crystal imaged at intervals of 0.9 s revealing different localization of individual cellulases. (B) Time dependence from the initial position of individual cellulase positions when acting on cellulose crystal as deduced by image processing of HS-AFM topographs. The colored symbols reflect data extracted from individual enzymes whereas the dotted line depict an average velocity as reported (Igarashi et al., 2009). Reprinted with permission from Igarashi et al. (2011). Copyright (2011) AAAS.

Mentions: One intriguing example of the application of high speed AFM to EPS is the characterization of cellulase induced degradation of crystalline cellulose specimen (Igarashi et al., 2011). Although the employed cellulose Iα crystal was obtained from processing of a sample from green alga Cladohora sp. is not a truly microbial EPS, the similarity of the specimen with bacterial cellulose is considered sufficiently close to include this example here. This cellulose crystal specimen was incubated on a graphite sample disk, and following addition of cellulase Cel7A from Trichoderma reesei, in buffered solution, sequential AFM imaging was performed at frame rates of 1–4 Hz. The movement of the Cel7A was observed only on the top of the crystalline cellulose specimen. Example data from their seminal report (Figure 3) shows relocation of individual cellobiohydrolase enzymes unidirectionally along the crystalline specimen, with a migration speed that is uneven and differs between the individual enzymes, but spread around the mean value as estimated from other average data. The data also revealed that individual enzymes does not exhibit a constant migration velocity, some enzymes were migrating without stopping within the observation areas, whereas others were apparently halting, suggested to arise from the Cel7A molecules exhibiting intermittently halt and go movements. The results were also discussed in terms of Cel7A interacting with the cellulose crystal in a productive and non-productive adsorption mode. Furthermore, detailed analysis of the dynamics revealed congestion between individual cellulases when moving on the crystalline specimen. As part of this, the inability of a particular enzyme to circumvent a stalled enzyme on the crystalline specimen was identified. Detailed molecular studies using high speed AFM imaging, such as that highlighted above, provide additional microscopic details on the statistical nature and congestion effects on the bioconversion of cellulose crystals that are not easily captured by other approaches. Application of HS-AFM in biological systems has recently been reviewed (Ando et al., 2014).


Novel imaging technologies for characterization of microbial extracellular polysaccharides.

Lilledahl MB, Stokke BT - Front Microbiol (2015)

High speed AFM imaging of Trocoderma reesei Cel7A cellulase acting on crystalline Iα cellulose. (A) AFM topographs of the same area of the cellulose crystal imaged at intervals of 0.9 s revealing different localization of individual cellulases. (B) Time dependence from the initial position of individual cellulase positions when acting on cellulose crystal as deduced by image processing of HS-AFM topographs. The colored symbols reflect data extracted from individual enzymes whereas the dotted line depict an average velocity as reported (Igarashi et al., 2009). Reprinted with permission from Igarashi et al. (2011). Copyright (2011) AAAS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446548&req=5

Figure 3: High speed AFM imaging of Trocoderma reesei Cel7A cellulase acting on crystalline Iα cellulose. (A) AFM topographs of the same area of the cellulose crystal imaged at intervals of 0.9 s revealing different localization of individual cellulases. (B) Time dependence from the initial position of individual cellulase positions when acting on cellulose crystal as deduced by image processing of HS-AFM topographs. The colored symbols reflect data extracted from individual enzymes whereas the dotted line depict an average velocity as reported (Igarashi et al., 2009). Reprinted with permission from Igarashi et al. (2011). Copyright (2011) AAAS.
Mentions: One intriguing example of the application of high speed AFM to EPS is the characterization of cellulase induced degradation of crystalline cellulose specimen (Igarashi et al., 2011). Although the employed cellulose Iα crystal was obtained from processing of a sample from green alga Cladohora sp. is not a truly microbial EPS, the similarity of the specimen with bacterial cellulose is considered sufficiently close to include this example here. This cellulose crystal specimen was incubated on a graphite sample disk, and following addition of cellulase Cel7A from Trichoderma reesei, in buffered solution, sequential AFM imaging was performed at frame rates of 1–4 Hz. The movement of the Cel7A was observed only on the top of the crystalline cellulose specimen. Example data from their seminal report (Figure 3) shows relocation of individual cellobiohydrolase enzymes unidirectionally along the crystalline specimen, with a migration speed that is uneven and differs between the individual enzymes, but spread around the mean value as estimated from other average data. The data also revealed that individual enzymes does not exhibit a constant migration velocity, some enzymes were migrating without stopping within the observation areas, whereas others were apparently halting, suggested to arise from the Cel7A molecules exhibiting intermittently halt and go movements. The results were also discussed in terms of Cel7A interacting with the cellulose crystal in a productive and non-productive adsorption mode. Furthermore, detailed analysis of the dynamics revealed congestion between individual cellulases when moving on the crystalline specimen. As part of this, the inability of a particular enzyme to circumvent a stalled enzyme on the crystalline specimen was identified. Detailed molecular studies using high speed AFM imaging, such as that highlighted above, provide additional microscopic details on the statistical nature and congestion effects on the bioconversion of cellulose crystals that are not easily captured by other approaches. Application of HS-AFM in biological systems has recently been reviewed (Ando et al., 2014).

Bottom Line: Understanding of biology is underpinned by the ability to observe structures at various length scales.This is so in a historical context and is also valid today.Evolution of novel insight often emerges from technological advancement.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology Trondheim, Norway.

ABSTRACT
Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects.

No MeSH data available.


Related in: MedlinePlus