Middleton, WI, United States
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Mead D.,Lucigen Corporation | Mead D.,C5-6 Technologies, Inc. | Mead D.,University of Wisconsin - Madison | Drinkwater C.,Lucigen Corporation | And 3 more authors.
PLoS ONE | Year: 2013

Background: Alkaliphilic Bacillus species are intrinsically interesting due to the bioenergetic problems posed by growth at high pH and high salt. Three alkaline cellulases have been cloned, sequenced and expressed from Bacillus cellulosilyticus N-4 (Bcell) making it an excellent target for genomic sequencing and mining of biomass-degrading enzymes. Methodology/Principal Findings: The genome of Bcell is a single chromosome of 4.7 Mb with no plasmids present and three large phage insertions. The most unusual feature of the genome is the presence of 23 LPXTA membrane anchor proteins; 17 of these are annotated as involved in polysaccharide degradation. These two values are significantly higher than seen in any other Bacillus species. This high number of membrane anchor proteins is seen only in pathogenic Gram-positive organisms such as Listeria monocytogenes or Staphylococcus aureus. Bcell also possesses four sortase D subfamily 4 enzymes that incorporate LPXTA-bearing proteins into the cell wall; three of these are closely related to each other and unique to Bcell. Cell fractionation and enzymatic assay of Bcell cultures show that the majority of polysaccharide degradation is associated with the cell wall LPXTA-enzymes, an unusual feature in Gram-positive aerobes. Genomic analysis and growth studies both strongly argue against Bcell being a truly cellulolytic organism, in spite of its name. Preliminary results suggest that fungal mycelia may be the natural substrate for this organism. Conclusions/Significance: Bacillus cellulosilyticus N-4, in spite of its name, does not possess any of the genes necessary for crystalline cellulose degradation, demonstrating the risk of classifying microorganisms without the benefit of genomic analysis. Bcell is the first Gram-positive aerobic organism shown to use predominantly cell-bound, non-cellulosomal enzymes for polysaccharide degradation. The LPXTA-sortase system utilized by Bcell may have applications both in anchoring cellulases and other biomass-degrading enzymes to Bcell itself and in anchoring proteins other Gram-positive organisms. © 2013 Mead et al.


Brumm P.,C5-6 Technologies, Inc. | Land M.L.,Oak Ridge National Laboratory | Hauser L.J.,Oak Ridge National Laboratory | Jeffries C.D.,Los Alamos National Laboratory | And 2 more authors.
Standards in Genomic Sciences | Year: 2015

Geobacillus sp. Y412MC52 was isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Based on 16S rRNA genes and average nucleotide identity, Geobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The genome of Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883 bp, an average G + C content of 52 % and one circular plasmid of 45,057 bp and an average G + C content of 45 %. Y412MC52 possesses arabinan, arabinoglucuronoxylan, and aromatic acid degradation clusters for degradation of hemicellulose from biomass. Transport and utilization clusters are also present for other carbohydrates including starch, cellobiose, and aα- and β-galactooligosaccharides. © 2015 Brumm et al.


Brumm P.,C5-6 Technologies, Inc. | Land M.L.,Oak Ridge National Laboratory | Hauser L.J.,Oak Ridge National Laboratory | Jeffries C.D.,Los Alamos National Laboratory | And 2 more authors.
Bioenergy Research | Year: 2015

Geobacillus thermoglucosidasius Y4.1MC1 was isolated from a boiling spring in the lower geyser basin of Yellowstone National Park. This species is of interest because of its metabolic versatility. The genome consists of one circular chromosome of 3,840,330 bp and a circular plasmid of 71,617 bp with an average GC content of 44.01 %. The genome is available in the GenBank database (NC_014650.1 and NC_014651.1). In addition to the expected metabolic pathways for sugars and amino acids, the Y4.1MC1 genome codes for two separate carbon monoxide utilization pathways, an aerobic oxidation pathway and an anaerobic reductive acetyl CoA (Wood-Ljungdahl) pathway. This is the first report of a non-anaerobic organism with the Wood-Ljungdahl pathway. This anaerobic pathway permits the strain to utilize H2 and fix CO2 present in the hot spring environment. Y4.1MC1 and its related species may play a significant role in carbon capture and sequestration in thermophilic ecosystems and may open up new routes to produce biofuels and chemicals from CO, H2, and CO2. © 2015, The Author(s).


PubMed | C5-6 Technologies, Inc., Lucigen Corporation, Oak Ridge National Laboratory and Los Alamos National Laboratory
Type: | Journal: Standards in genomic sciences | Year: 2015

Geobacillus sp. Y412MC52 was isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Based on 16S rRNA genes and average nucleotide identity, Geobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The genome of Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883bp, an average G+C content of 52% and one circular plasmid of 45,057bp and an average G+C content of 45%. Y412MC52 possesses arabinan, arabinoglucuronoxylan, and aromatic acid degradation clusters for degradation of hemicellulose from biomass. Transport and utilization clusters are also present for other carbohydrates including starch, cellobiose, and - and -galactooligosaccharides.


PubMed | C5-6 Technologies, Inc., Lucigen Corporation, Oak Ridge National Laboratory and Los Alamos National Laboratory
Type: | Journal: Standards in genomic sciences | Year: 2016

[This corrects the article DOI: 10.1186/s40793-015-0075-0.].


De Maayer P.,University of Pretoria | Brumm P.J.,C5-6 Technologies, Inc. | Mead D.A.,C5-6 Technologies, Inc. | Cowan D.A.,University of Pretoria
BMC Genomics | Year: 2014

Background: Members of the thermophilic genus Geobacillus can grow at high temperatures and produce a battery of thermostable hemicellulose hydrolytic enzymes, making them ideal candidates for the bioconversion of biomass to value-added products. To date the molecular determinants for hemicellulose degradation and utilization have only been identified and partially characterized in one strain, namely Geobacillus stearothermophilus T-6, where they are clustered in a single genetic locus. Results: Using the G. stearothermophilus T-6 hemicellulose utilization locus as genetic marker, orthologous hemicellulose utilization (HUS) loci were identified in the complete and partial genomes of 17/24 Geobacillus strains. These HUS loci are localized on a common genomic island. Comparative analyses of these loci revealed extensive variability among the Geobacillus hemicellulose utilization systems, with only seven out of 41-68 proteins encoded on these loci conserved among the HUS+ strains. This translates into extensive differences in the hydrolytic enzymes, transport systems and metabolic pathways employed by Geobacillus spp. to degrade and utilize hemicellulose polymers. Conclusions: The genetic variability among the Geobacillus HUS loci implies that they have variable capacities to degrade hemicellulose polymers, or that they may degrade distinct polymers, as are found in different plant species and tissues. The data from this study can serve as a basis for the genetic engineering of a Geobacillus strain(s) with an improved capacity to degrade and utilize hemicellulose. © 2014 De Maayer et al.; licensee BioMed Central Ltd.


Brumm P.J.,C5-6 Technologies, Inc. | Brumm P.J.,U.S. Department of Energy
Biofuels | Year: 2013

Genome sequencing of cellulolytic bacteria combined with analyses using structural and sequence similarity software reveals potential cellulolytic enzymes not previously recovered by routine purification or shotgun cloning techniques. Comparison of the presence and absence of potential cellulases across the prokaryotes indicate that there are at least four distinct mechanisms of cellulose degradation; two distinct soluble systems and two separate cell-based systems can be postulated. None of the mechanisms appear completely homologous to the Trichoderma system of cellulose degradation. Minimum sets of bacterial cellulases can be predicted for the soluble sets based on comparison across genomes, leading to testable hypotheses on cellulose degradation. © 2013 Future Science Ltd.


Patent
C5-6 Technologies, Inc. | Date: 2013-11-11

Enzymes for inhibiting growth of biofilms and degrading biofilms. The enzymes comprise glycosyl hydrolases capable of degrading biofilms. The enzymes are formulated in compositions with and without antimicrobial agents. The enzymes with and without the antimicrobial agents are delivered to biofilms to degrade the biofilms and treat infections of microorganisms associated with the biofilms, delivered to surfaces to inhibit growth of biofilms thereon, and administered to animals to inhibit growth of biofilms therein.


C5-6 Technologies, Inc. | Entity website

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Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 227.83K | Year: 2011

DESCRIPTION (provided by applicant): Improved therapy has transformed cystic fibrosis (CF) from a disease characterized by death in early childhood to a chronic illness with a median survival of approximately 30 years. However, there is still no cure for this devastating genetic disease affecting approximately 30,000 Americans and many questions about molecular pathogenesis, with important implications for therapy, remain to be answered. Approximately 80% of cystic fibrosis patients develop Pseudomonas aeruginosa infections of the lungs, which are a major cause of morbidity and mortality in this genetic disease. These chronic infections are resistant to antibiotic therapies. Recent studies suggest that the antibiotic resistance of P. aeruginosa in the CF lung is due to the formation of biofilms that are impervious to antibiotics. The P. aeruginosa biofilm is composed of bacterial cells surrounded by a three dimensional matrix of diverse substances, including polysaccharides, proteins, nucleic acids, and lipids; the major structural component appears to be polysaccharides produced by the P. aeruginosa cells. The proposed work will systematically test the ability of 80 polysaccharide-degrading enzymes to breakdown or weaken the P. aeruginosa biofilm. A sensitivetest to determine the amount of polysaccharide degradation has been developed and those enzymes that actively degrade the biofilm will be combined to make a synergistic enzyme cocktail. The optimal enzyme mixture will then be tested in combination with Tobramycin to test the hypothesis that enzymatic degradation of the P. aeruginosa polysaccharide biofilm components will increase the sensitivity of P. aeruginosa to antibiotic treatment, resulting in improved outcomes for patients. PUBLIC HEALTH RELEVANCE: There is an unmet need for new therapies to extend the life and health of cystic fibrosis patients, a fatal disease affecting tens of thousands of Americans. Pseudomonas aeruginosa is the pathogen responsible for most CF patients succumbing to thisgenetic disease. The proposed development of enzymes that attack and degrade the antibiotic resistant biofilm formed by Pseudomonas aeruginosa has the potential to extend the quality and quantity of life, with important implications for numerous other diseases that are impacted by the formation of biofilms.

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