High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Abstract

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.

Effects of dietary Bacillus cereus G19, B. cereus BC-01, and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression of immune-related genes in coelomocytes and intestine of the sea cucumber (Apostichopus japonicus Selenka)

“…However, B. cereus G19 and B. cereus BC-01 supplementation had no significant effect on the transcriptional level of the Aj-p105, Aj-p50, and Aj-rel genes in the mid-intestine tissue compared to those in the control group. Dobrijevic et al [65] reported that B. subtilis VI7695, a grampositive model bacterium, played a neutral in the NF-kB activation assay in human HT-29 intestine epithelial cells. Similarly, our results might also indicate that dietary B. cereus G19 and B. cereus BC-01 supplementation play a neutral role in the activation of NFkB in the intestine of sea cucumber.…”

β‐Glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus

“…Thus, L. lactis MG1363 appears to be quite limited in its ability to successfully express genes from other organisms, and alternative Gram‐positive hosts (e.g. Bacillus subtilis , Dobrijevic et al ., ) may be more suitable for future functional metagenomic studies. This study does, however, also show the limitations in using E. coli as heterologous host, as the clone expressing very high activity in L. lactis MG1363 (P20A8) did not exhibit good activity in E. coli XL1 Blue.…”

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining

“…The first of these is the expression of soluble proteins. To overcome this problem, high throughput cloning and expression methods are being developed (Dobrijevic et al , 2013, Groisillier et al, 2010, Vincentelli et al, 2011. A second challenge will be to acquire better understanding of the role of individual enzymes with the biocatalytic diversity produced by microbial communities.…”