Physiological Studies of Chlorobiaceae Suggest that Bacillithiol Derivatives Are the Most Widespread Thiols in Bacteria.

Low-molecular-weight (LMW) thiols mediate redox homeostasis and the detoxification of chemical stressors. Despite their essential functions, the distribution of LMW thiols across cellular life has not yet been defined. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae Here we show that Chlorobaculum tepidum synthesizes a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S, which suggests that the new LMW thiol is closely related to bacillithiol (BSH), the major LMW thiol of low-G+C Gram-positive bacteria. The Cba. tepidum LMW thiol structure was N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, the fourth instance of this modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome and the CT1040 gene product, N-Me-BSH synthase, were required for N-Me-BSH synthesis. N-Me-BSH was found in all Chlorobiaceae examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobiaceae, Bacteroidetes, Deinococcus-Thermus, and Firmicutes but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Thus, BSH and derivatives appear to be the most broadly distributed LMW thiols in biology.IMPORTANCE Low-molecular-weight thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new thiol, N-methyl-bacillithiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacillithiol, the main thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacillithiol and its derivatives are the most widely distributed thiol metabolites in biology.

A bacterial pioneer produces cellulase complexes that persist through community succession.

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, 'Candidatus Reconcilibacillus cellulovorans', possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the 'Ca. Reconcilibacillus cellulovorans' multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger.

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the ß-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis.

UNLABELLED: Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCE: Cellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.

Refining the phylum Chlorobi by resolving the phylogeny and metabolic potential of the representative of a deeply branching, uncultivated lineage.

Recent studies have expanded the phylum Chlorobi, demonstrating that the green sulfur bacteria (GSB), the original cultured representatives of the phylum, are a part of a broader lineage whose members have more diverse metabolic capabilities that overlap with members of the phylum Bacteroidetes. The 16S rRNA gene of an uncultivated clone, OPB56, distantly related to the phyla Chlorobi and Bacteroidetes, was recovered from Obsidian Pool in Yellowstone National Park; however, the detailed phylogeny and function of OPB56 and related clones have remained unknown. Culturing of thermophilic bacterial consortia from compost by adaptation to grow on ionic-liquid pretreated switchgrass provided a consortium in which one of the most abundant members, NICIL-2, clustered with OPB56-related clones. Phylogenetic analysis using the full-length 16S rRNA gene from NICIL-2 demonstrated that it was part of a monophyletic clade, referred to as OPB56, distinct from the Bacteroidetes and Chlorobi. A near complete draft genome (>95% complete) was recovered from metagenomic data from the culture adapted to grow on ionic-liquid pretreated switchgrass using an automated binning algorithm, and this genome was used for marker gene-based phylogenetic analysis and metabolic reconstruction. Six additional genomes related to NICIL-2 were reconstructed from metagenomic data sets obtained from thermal springs at Yellowstone National Park and Nevada Great Boiling Spring. In contrast to the 16S rRNA gene phylogenetic analysis, protein phylogenetic analysis was most consistent with the clustering of the Chlorobea, Ignavibacteria and OPB56 into a single phylum level clade. Metabolic reconstruction of NICIL-2 demonstrated a close linkage with the class Ignavibacteria and the family Rhodothermaceae, a deeply branching Bacteroidetes lineage. The combined phylogenetic and functional analysis of the NICIL-2 genome has refined the membership in the phylum Chlorobi and emphasized the close evolutionary and metabolic relationship between the phyla Chlorobi and the Bacteroidetes.

High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal ß-glucosidase production.

Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS)-based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for ß-glucosidase production under various growth conditions. Media that produced high ß-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC), Medium 84 + rolled oats, and M9TE + MCC at 45°C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45°C than at all other temperatures. While T. bispora is reported to grow optimally at 60°C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45°C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

Sulfite oxidation in chlorobaculum tepidum.

The green sulfur bacterium Chlorobaculum tepidum is proposed to oxidize sulfide and elemental sulfur via sulfite as an obligate intermediate. The sulfite pool is predicted to be contained in the cytoplasm and be oxidized by the concerted action of ApsBA, which directly oxidizes sulfite, and QmoABC, which transfers electrons from ApsBA to the quinone pool. Like other green sulfur bacteria, C. tepidum was unable to use exogenously provided sulfite as the sole electron donor. However, exogenous sulfite significantly stimulated the growth yield of sulfide limited batch cultures. The growth of C. tepidum mutant strains, CT0867/qmoB::TnOGm and CT0868/qmoC::TnOGm, was not increased by sulfite. Furthermore, these strains accumulated sulfite and displayed a growth yield decrease when grown on sulfide as the sole electron donor. These results support an obligate, cytoplasmic sulfite intermediate as part of the canonical sulfur oxidation pathway in C. tepidum that requires the Qmo complex for oxidation.

Beyond the genome: functional studies of phototrophic sulfur oxidation.

The increasing availability of complete genomic sequences for cultured phototrophic bacteria and assembled metagenomes from environments dominated by phototrophs has reinforced the need for a "post-genomic" analytical effort to test models of cellular structure and function proposed from genomic data. Comparative genomics has produced a testable model for pathways of sulfur compound oxidation in the phototrophic bacteria. In the case of sulfide, two enzymes are predicted to oxidize sulfide: sulfide:quinone oxidoreductase and flavocytochrome c sulfide dehydrogenase. However, these models do not predict which enzyme is important under what conditions. In Chlorobaculum tepidum, a model green sulfur bacterium, a combination of genetics and physiological analysis of mutant strains has led to the realization that this organism contains at least two active sulfide:quinone oxidoreductases and that there is significant interaction between sulfide oxidation and light harvesting. In the case of elemental sulfur, an organothiol intermediate of unknown structure has been proposed to activate elemental sulfur for transport into the cytoplasm where it can be oxidized or assimilated, and recent approaches using classical metabolite analysis have begun to shed light on this issue both in C. tepidum and the purple sulfur bacterium Allochromatium vinosum.