Nanoscale resolution of microbial fiber degradation in action
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
The premise behind this manuscript is timely and of interest to a broad scientific community working in the field of microbial recycling of cellulosic biomass. It provides a useful link between the occurrence and molecular aspects of the bacterial 'machinery' named cellulosome, and physiological traits of the same bacteria when grown on micro-crystalline cellulose. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical Clostridium thermocellum system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form ‘cellulosome capsules’ driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.
Article activity feed
-
-
Evaluation Summary:
The premise behind this manuscript is timely and of interest to a broad scientific community working in the field of microbial recycling of cellulosic biomass. It provides a useful link between the occurrence and molecular aspects of the bacterial 'machinery' named cellulosome, and physiological traits of the same bacteria when grown on micro-crystalline cellulose. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
-
Reviewer #1 (Public Review):
The manuscript by Tatli et al. entitled "Nanoscale resolution of microbial fiber degradation in action" characterizes how the cellulosome-producing, anaerobic bacterium Clostridium thermocellum responds to the presence of crystalline cellulose substrate and its subsequent degradation in real-time. Using state-of-the-art cryo-electron structural methods (i.e., microscopy and tomography) in combination with biochemistry, molecular biology, imaging, and microbial genetics and physiology the authors assess the location, density, enzyme composition of the cellulosomal complexes on the bacterial surface, its interactions with the crystalline cellulose substrate, and the corresponding changes in these properties that result from decomposition the substrate over time.
Specifically, using cryo-electron-based methods …
Reviewer #1 (Public Review):
The manuscript by Tatli et al. entitled "Nanoscale resolution of microbial fiber degradation in action" characterizes how the cellulosome-producing, anaerobic bacterium Clostridium thermocellum responds to the presence of crystalline cellulose substrate and its subsequent degradation in real-time. Using state-of-the-art cryo-electron structural methods (i.e., microscopy and tomography) in combination with biochemistry, molecular biology, imaging, and microbial genetics and physiology the authors assess the location, density, enzyme composition of the cellulosomal complexes on the bacterial surface, its interactions with the crystalline cellulose substrate, and the corresponding changes in these properties that result from decomposition the substrate over time.
Specifically, using cryo-electron-based methods and imaging the authors showed extracellular cellulosomal densities at resolutions not seen previously and were able to measure distances between the bacterial S-layer and the cellulosome layer as well of the thickness of the latter. Taking advantage of cryo-electron tomography methods and data processing, the authors present nano-scale images of cellulosome-crystalline cellulose interactions, where the cellulosomal machinery is seen to envelop the substrate and disrupt the well-order compact, packing of cellulose microfibrils. They also present the cryo-EM structure of Cel48S, the most abundant cellulosomal glycoside hydrolase, which had a similar fold to the catalytic module previously determined by X-ray crystallography but also the topology of the linker region tethering the catalytic module to its type-I dockerin module that had not been previously observed. Expression of Cel48S in C. thermocellum was dramatically increased upon exposure to the crystalline cellulose substrate for the first 10-15 hr after which there was a subsequent decrease to basal levels between hours 15-20, which was associated with substrate availability and increased presence of degradation bioproducts. Finally, the authors used cryo-electron microscopy to assess single-cell cellulosome distribution across the bacterial population and its substrate dependency. Rather than a distribution of cellulosome densities on the cell surface across the microbial population, two predominant phenotypes were observed - a high-density phenotype and a low-density phenotype that shifted from a 1:5 to 5:1 ratio upon exposure to cellulose. The authors associate these latter observations with division-of-labour and bet-hedging evolutionary strategy whereby a population invested significant energy to produce the cellulosomal machinery and are thus primed for a substrate-rich environment while the low-density population ensures continued cell growth and nimble response to changing environmental conditions.
Strengths:
The manuscript is well-written and represents an influential body of work that will have broad appeal, including the environmental microbe, carbohydrate/biomass degradation, microbial and biopolymer engineering communities. The experiments are well-designed comprising ingenious use of microbial genetics, various substrates, and recombinant protein constructs with the C. thermocellum system to address observations in cyro-electron, biochemical and microbiology studies. The experimental analysis and interpretation are first-rate. The methods are appropriate, diverse yet truly complementary, and state-of-the-art.
While much effort has been dedicated to the structural characterization of the cellulosome, it has largely involved a dissection approach involving recombinant proteins. As such, there remains a significant gap in knowledge of the in vivo cellulosome structure and its interaction with crystalline cellulose. Furthermore, little is currently known as to how cellulosome-producing bacteria respond to changing environments, including C. thermocellum, which serves as the model cellulosomal bacterium. The data provides unprecedented in situ views of the cellulosomal machinery on the bacterial cell surface, its interaction of cellulose, and the disruption of the latter's structural organization. The quantitative nature of the work, particularly those associated with revealing the dynamic yet quite specific phenotypic heterogeneity of cellulosome-producing C. thermocellum (a high-density and a low-density population), is innovative in its approach and novel. These findings present intrigues and previously unforeseen insights into the response of C. thermocellum to the cellulose substrate. The authors have done an excellent job of linking previous observations to one made here using them to establish a foundation from which they formulate their conclusions.
Weaknesses:
There are no major weaknesses that significantly detract from the novelty and impact of the study. Not so much a weakness as much as simply unfortunate is the lack of the type-I dockerin module in the cryo-EM structure of Cel48S. The authors correctly note the apparent inherent flexibility of the N-terminal region of the dockerin module and the low calcium concentrations used, which may contribute to its absence in the structure.
-
Reviewer #2 (Public Review):
The manuscript by Itzhak Mizrahi is an original study using state-of-the-art integrative structural biology at multiple scales, using cryo-EM, imaging, electron tomography, microbiology and genetics to capture anaerobic bacterial cellulosomes from C. thermocellum in action. The study depicts the presence of cellulosomes at the bacterial extracellular surface, the impact of cellulosomal action on micro-crystalline cellulose, and identifies the presence of large globular enzymes in interaction with the substrate. Major findings are the multi-scale description of a 65 nm thick "belt" of cellulosomal particles around a single bacterial cell when displaying a high density of cellulosomes, down to the identification of specific components, such as the catalytic enzyme Cel48S, within this region when interacting …
Reviewer #2 (Public Review):
The manuscript by Itzhak Mizrahi is an original study using state-of-the-art integrative structural biology at multiple scales, using cryo-EM, imaging, electron tomography, microbiology and genetics to capture anaerobic bacterial cellulosomes from C. thermocellum in action. The study depicts the presence of cellulosomes at the bacterial extracellular surface, the impact of cellulosomal action on micro-crystalline cellulose, and identifies the presence of large globular enzymes in interaction with the substrate. Major findings are the multi-scale description of a 65 nm thick "belt" of cellulosomal particles around a single bacterial cell when displaying a high density of cellulosomes, down to the identification of specific components, such as the catalytic enzyme Cel48S, within this region when interacting and degrading cellulose micro-fibrils. These single-cell data are put in perspective with physiological growth properties of native and genetically modified C. thermocellum, showing that the bacterial population is heterogeneous with respect to the presence of cellulosomal complexes. Two types of populations, one displaying high density and a second with a much lower density of cellulosomes, co-exist in a ratio that depends on the available monosaccharides in solution, leading the authors to speculate that a division-of-labor strategy takes place in the C. thermocellum population.
The conclusions drawn are clearly justified by the presented data, interpretations and even speculations are designed as such by the authors and are plausible in view of the obtained results. The strength of the paper is the clever application of methods that allow spanning scales by several orders of magnitude, and that allow connecting single-cell data to physiological observations in the bulk of cultured cells.
-
