Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis

Abstract

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.

Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome

“…The Bacteroides, the most abundant genus within the gut of US residents (The Human Microbiome Project, 2012; Yatsunenko et al, 2012), are capable of utilizing both dietary and host-derived nutrient sources (Martens et al, 2008; Sonnenburg et al, 2005), are known to have an important role in immune development (Dasgupta et al, 2014; Mazmanian et al, 2005; Sefik et al, 2015), and possess a vast array of sensors relevant to the gut environment (Xu et al, 2003). Although tools are available for genetic manipulation (Goodman et al, 2009; Koropatkin et al, 2008; Wang et al, 2000) and expression in Bacteroides (Mastropaolo et al, 2009; Mimee et al, 2015; Parker and Jeffrey Smith, 2012; Smith et al, 1992; Whitehead and Hespell, 1990), genomic integrations are limited in throughput (several at one time) and laborious (>7 days) due to the requirement of bacterial conjugation. Even the strongest promoters identified to date are insufficient for in vivo microscopic imaging of fluorescent protein expression (Mimee et al, 2015).…”

Xylanolytic activities ofSpirochaeta thermophila

“…The organism possesses the major xylan-degrading enzyme activities, namely xylanase, xylosidase, and arabinosidase ( Table 3). [24,25]. Xylanolytic activities were highly expressed with S. thermophila growth on xylan, and significant…”

Utilization of xylooligosaccharides by selected ruminal bacteria

“…Presumably, these organisms generate oligomeric products as intermediates during the normal degradation of xylan. Indeed, it has been shown that the endoxylanase from P. ruminicola hydrolyzes xylan to xylotriose and oligosaccharides with greater degrees of polymerization (25). What is interesting are some insights that these observations provide into the xylan-utilizing abilities and limitations of these organisms.…”