Dataset Information

Unraveling essential cellulosomal components of the (Pseudo)Bacteroides cellulosolvens reveals an extensive reservoir of novel catalytic enzymes.

ABSTRACT:

Background

(Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium.

Results

The current proteome-wide work reveals patterns of protein expression of the various cellulosomal components, and explores the signature of differential expression upon growth of the bacterium on two major carbon sources-cellobiose and microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing components (mainly enzymes), in addition to free enzymatic subunits. The dockerin-bearing components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family members were represented among 102 carbohydrate-degrading enzymes, including the omnipresent, most abundant GH48 exoglucanase. Specific cellulosomal components were found in different molecular-weight fractions associated with cell growth on different carbon sources. Overall, microcrystalline cellulose-derived cellulosomes showed markedly higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different cellulosic and/or hemicellulosic carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by Clostridium thermocellum purified cellulosomes.

Conclusions

The current research demonstrates the involvement of key cellulosomal factors that participate in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies on various carbon sources was revealed. The novel reservoir of cellulolytic components of the cellulosomal degradation machineries may serve as a pool for designing new cellulolytic cocktails for biotechnological purposes.

SUBMITTER: Zhivin-Nissan O

PROVIDER: S-EPMC6507058 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Unraveling essential cellulosomal components of the (Pseudo)Bacteroides cellulosolvens reveals an extensive reservoir of novel catalytic enzymes.

Zhivin-Nissan Olga O Dassa Bareket B Morag Ely E Kupervaser Meital M Levin Yishai Y Bayer Edward A EA

Biotechnology for biofuels 20190509

<h4>Background</h4>(Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of th ...[more]

PMID: 31086567

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Project description:Background (Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium producing the most extensive and intricate cellulosomal system known in nature. Recently, an elaborate architecture of B. cellulosolvens cellulosomal system was revealed from its genome sequence analysis, and first evidence on the interactions between its structural and enzymatic components were detected in vitro. Yet, the cellulolytic potential of the bacterium in carbohydrate deconstruction may reside only within complete high-molecular weight protein complexes, which are secreted from the bacterium. Results The current proteome-wide work reveals patterns of protein expression of various cellulosomal components, and explores the differential expression signature upon bacterial growth two carbon sources, either cellobiose or microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome fraction revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing enzymes, in addition to free enzymatic subunits. All these components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family memebers were represented among 102 carbohydrate-degrading enzymes, including the most abundant GH48 exoglucanase. Specific cellulosomal components were associatred with different carbon sources, in cellulosomal fractions of different molecular weights. Overall, microcrystalline cellulose-derived cellulosomes showed higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by C. thermocellum purified cellulosomes. Conclusions The current research demonstrates the involvement of key cellulosomal factors participating in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies of various carbon sourcesis revealed. Thus, a novel components reservoir of degradation machineries that may serve for subsequent cellulosomal research, as a pool for designing new cellulolytic cocktails for biotechnological purposes.

Project description:Background(Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature.ResultsThe organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin-dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin-dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes).ConclusionsThe results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin-dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

Project description:Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.

Project description:BackgroundDiet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.ResultsTranscriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70%) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.ConclusionThis study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.

Dataset Information

Unraveling essential cellulosomal components of the (Pseudo)Bacteroides cellulosolvens reveals an extensive reservoir of novel catalytic enzymes.

Background

Results

Conclusions

Publications

Unraveling essential cellulosomal components of the (<i>Pseudo</i>)<i>Bacteroides cellulosolvens</i> reveals an extensive reservoir of novel catalytic enzymes.

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