Expression of Cellulosome Components and Type IV Pili within the Extracellular Proteome of Ruminococcus flavefaciens 007

Maša Vodovnik,Martin D Reid,Margret E Berg Miller,Harry J Flint,Louise Cantlay,Bryan A White,Julian Parkhill,Sylvia H Duncan,Carl J Yeoman,Keith Turner,Edward A Bayer,Romana Marinšek-Logar,Raphael Lamed

doi:10.1371/journal.pone.0065333

Abstract

Background Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose.Methodology/Principal FindingsThe major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel.Conclusions/SignificanceThis study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

Highlights

Bacteria related to Ruminococcus flavefaciens play an important role in the degradation of lignocellulosic plant fibre in the rumen and large intestine of herbivorous animals
Ruminococcus flavefaciens 007 was first isolated from rumen fluid as an actively cellulolytic culture able to degrade dewaxed cotton cellulose
Strain 007S cannot grow on de-waxed cotton cellulose, but its growth with cellobiose, Avicel and oat spelt xylan is similar to that of 007C (Fig. S1, [9])

Summary

Introduction

Bacteria related to Ruminococcus flavefaciens play an important role in the degradation of lignocellulosic plant fibre in the rumen and large intestine of herbivorous animals. Many R. flavefaciens strains isolated from the rumen are able to degrade insoluble crystalline cellulose present in test substrates such as filter paper and dewaxed cotton fibre. Earlier work reported a mutational variant of R. flavefaciens strain 007C (referred to as 007S) that had lost the ability to degrade dewaxed cotton cellulose, while largely retaining the ability to degrade other forms of insoluble cellulose such as Avicel [9,10]. This difference was thought likely to reflect differences in substrate adhesion resulting from changes in extracellular protein expression that potentially identify the key proteins involved [8]. This has allowed us to investigate the factors required for degradation of this recalcitrant form of cellulose

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