Real-Time Visualization of Cellulase Activity by Microorganisms on Surface

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root–microorganism interactions.

Systematic Analysis of Escherichia coli Isolates from Sheep and Cattle Suggests Adaption to the Rumen Niche

ABSTRACT The commonly used laboratory bacterium Escherichia coli normally does not produce and secrete cellulases due to its complex bilayer membrane structure and poor secretory apparatus. In our previous study, the cellulolytic E. coli strain ZH-4 with extracellular cellulase activity was found in the bovine rumen. In this study, we demonstrate that the secretion of cellulase is a common feature of E. coli isolates from the rumen of animals such as sheep and cattle. Physiological phenotype characterization of these E. coli isolates, together with genome, transcriptome, and comparative genomics analysis, suggests their adaption to the rumen niche. The higher growth rate of the isolated strains under aerobic conditions meets the competitive requirements of the strains in rumen microecosystem, while anaerobic accumulation of reduced H2 and succinate is hypothesized to be the results of adaptation to the rumen environment. Cellulase secretion increased significantly when the molecular chaperone genes ibpA and ibpB were overexpressed. This was also revealed by the transcriptomic data. A possible mechanism for cellulase secretion by E. coli isolates was proposed based on the transcriptomic data and molecular experiments. IMPORTANCE As an important intestinal microorganism, E. coli is present in the intestinal tract of animals and in many other environments. However, it normally does not produce and secret cellulases due to its complex bilayer membrane structure and poor secretory apparatus. Here, we proved that E. coli is widely present in the rumen of sheep and cattle. Systematic analysis of the isolates indicated that they have adapted to the rumen niche, with phenotypes that include secretion of cellulase and fermentative accumulation of succinate and H2. The finding that overexpression of small heat shock protein genes ibpA and ibpB could facilitate cellulase BcsZ secretion, which provides a possible insight into the protein secretion mechanism of rumen-colonizing E. coli.