Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.
This review presents an outline of our current understanding of ruminal nitrogen metabolism from three perspectives: proteolytic microorganisms and their enzymes, intraruminal recycling of microbial protein, and enzymes of ammonia assimilation. Some of the pending advances and future research opportunities in these areas are also discussed. The 'smugglin' concept appears to offer the potential to inhibit peptide-utilizing bacteria selectively in the rumen, as demonstrated by initial studies with Prevotella ruminicola. The relative contributions of protozoa-, bacteriophage-, and self-mediated lysis of bacteria to intraruminal recycling of microbial protein are not yet quantified, and further efforts to understand the biology and dynamics of ruminal bacteriophage and protozoa populations are warranted. In Ruminococcus flavefaciens and Prevotella ruminicola, glutamate dehydrogenase (GDH) appears to be the predominant route of ammonia assimilation irrespective of ammonia concentration, and peptides modulate GDH activity in P. ruminicola. The physiological basis behind the difference between optimal ammonia concentrations for ruminal fibre digestion and microbial protein synthesis remains unclear. Molecular biology techniques extend beyond their application in pursuit of the 'superbug' concept, by offering new and exciting opportunities to understand better microbial physiology, diversity, and ecology. Fundamental research in these areas must be continued if further advances in feed utilization and nutrient retention are to be realized.
G.T. HOWARD, J. VICKNAIR AND R.I. MACKIE. 2001. Aims: A plate assay to screen and detect bacterial polyurethanase in agar medium containing a colloidal polyester‐polyurethane and rhodamine B is presented. Methods and Results: Substrate hydrolysis causes the formation of orange fluorescent halos visible upon u.v. irradiation. The logarithm of polyurethanase activity from a purified polyurethanase protein is linearly correlated with the diameter of halos, thereby allowing quantification of polyurethanase activities ranging from 0·81 to 7·29 Units. Conclusions: The potential advantages of this system are in identification and recovery of viable polyurethanolytic bacteria and quantification of polyurethanase activity. Significance and Impact of the Study: These advantages are derived largely from the intense fluorescence observed due to the hydrolysis of substrate reacting with rhodamine B allowing for the use of low substrate concentrations and corresponding decrease in time required detecting low levels of enzyme activity.
Aims: To better understand the role of PueA and PueB from Pseudomonas chlororaphis in polyurethane degradation, the present study was conducted to create insertional mutants in their respective genes. Methods and Results: Growth kinetic studies showed that the pueA knockout mutant had a greater effect than the pueB knockout mutant. The pueA mutant had an 80% decrease in cell density from that of the wild type, while the pueB mutant had an 18% decrease in cell density. Polyurethane utilization followed Michaelis-Menten kinetics. The pueA and pueB mutants exhibited a 17% and 10% decrease respectively in growth rate using polyurethane when compared with the wild type. Conclusions: In this present study, pueA and pueB, are shown to be part of an ABC transporter gene cluster that consists of seven open reading frames. Mutational analysis results suggest that PueA may play a more major role in polyurethane degradation than PueB based on cell density and growth rates. Significance and Impact of the Study: The results from this study provide a starting point for the eventual enhancement and bioremediation of polyurethane waste. Understanding the role of polyurethane-degrading enzymes is useful for the creation of strains for this purpose.
Current knowledge of the uptake and metabolism of the major energy yielding and nitrogenous nutrients that are naturally available to ruminal bacteria is reviewed. The potential use of metabolic engineering to manipulate these metabolic pathways and improve nutrient utilization in ruminant animals is briefly discussed. Metabolic engineering is the use of recombinant DNA techniques to enhance microbial function by manipulating enzymatic, transport and regulatory functions of the cell. Examples of the use of metabolic engineering in industrial fermentation are also given.
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