Some (bacilli) like it hot: Genomics of Geobacillus species

Copyright © 2014 The Author. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.What are Geobacillus?BBSR

Isolation, genome sequencing, assembly and annotation of THERMOPHILIC Geobacillus thermoleovorans CCB_US3_UF5 from Ulu Slim Hot Spring, Perak, Malaysia.

Satu bakteria termofilik Geobacillus thermoleovorans CCB_US3_UF5 telah dipencil dari kolam air panas Ulu Slim, Perak, Malaysia. Untuk memahami kemandirian hidup G. thermoleovorans CCB_US3_UF5 memerlukan pengetahuan genomnya sebagai pelan perancangan adaptasi terhadap persekitaran panas. Penggunaan teknologi penjujukan generasi terkini telah digunakan untuk penjujukan genom bakteria. A thermophilic bacterium Geobacillus thermoleovorans CCB_US3_UF5 was isolated from Ulu Slim hot spring located in Perak, Malaysia. Understanding the survival of G. thermoleovorans CCB_US3_UF5 requires knowledge of its genome as the blueprint for high temperature adaptation to the environment. Genome sequencing of the bacterium was performed using next generation sequencing technology

<em>Geobacillus</em> Bacteria: Potential Commercial Applications in Industry, Bioremediation, and Bioenergy Production

The genus Geobacillus is represented by obligately thermophilic bacteria able to grow in the temperature range of 35–75°C. They are modest bacteria isolated from various sources on routine media such as nutrient agar. Originally classified as representatives of Bacillus, the species of Geobacillus were established in 2001 as a new genus. However, sequence similarity between all species indicates that at least some species need to be reclassified at the genus level. In addition to 16S rRNA, housekeeping genes, 16S-23S rRNA gene internal transcribed spacer, and repetitive sequences can be used in classification and identification of thermophilic bacteria. The ability to survive and grow at high temperatures as well as utilization and synthesis of a wide range of compounds makes these bacteria and their products attractive for use in various spheres: food, paper, biotechnology industries, medicine, bioremediation, etc. A broad spectrum of applications arouses increased interest in the study of physiological and biochemical characteristics and triggers emergence of new usage areas for Geobacillus, such as bioenergy. The growing demand for energy leads to the development of alternative technologic options. Geobacillus species demonstrated the ability to generate or enhance productivity of important sources of bioenergy such as ethanol, isobutanol, 2,3-butanediol, biodiesel, and biogas

Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing

The growing demand for consumables and energy, combined with increasing consciousness over environmental issues like global warming, faces us with the challenge to find alternatives for fossil resources. Alternative production methods for energy, like windmills, solar panels and hydroelectricity plants, are far developed and have become economically competitive to fossil resourcebased production processes. However, the production of many (bulk) chemicals and products is still dominated by the petroleum industry. One such chemical is lactic acid, a fermentation product of many bacteria and a compound that is gaining interest as a building block for poly lactic acid (PLA). PLA is a polymer used to produce bioplastics, and thereby provides an alternative to petroleumbased plastic production. As described in Chapter 1, economically feasible production of lactic acid is envisioned through consolidated bioprocessing (CBP). In a CBP process, pretreated lignocellulosic biomass is hydrolyzed to fermentable sugars and those sugars are subsequently fermented to desired product in one reaction vessel. The organism of choice for this hydrolyzation and fermentation is preferentially a thermophile, capable of enzyme production and lactic acid fermentation. Species from the genus Geobacillus have many of the desired characteristics, and in Chapter 2 we have enriched and isolated facultative anaerobic (hemi)cellulolytic Geobacillus strains from compost samples. By selecting for growth on both cellulose and xylan, 94 strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible with a transformation efficiency of 1.7×105 CFU/µg of plasmid DNA. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, we demonstrated that strain T12 is capable of direct conversion of beechwood xylan to mainly lactic acid in minimal media. Chapter 3 describes the genome sequencing and several features of G. thermodenitrificans T12. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 kb and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes is similar to those of the denitrification pathways found in strain NG80-2. The host-defence systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system that could potentially be exploited as a genome editing tool for thermophiles. Furthermore, the hemicellulose utilisation (HUS) locus of strain T12 appeared to have orthologues for all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. The HUS-locus associated gene, GtxynA1, encodes an extracellular endoxylanase of strain T12, and belongs to the family 10 glycoside hydrolases (GH10). In Chapter 4, we describe the cloning, expression and characterization of GtXynA1. The recombinant endoxylanase was purified to homogeneity and showed activity between 40°C and 80°C, with an optimum activity at 60°C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60°C. Highest activity was demonstrated towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 can degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endoxylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to be a potential candidate for CBP but lacks the desired endo and exoglucanases needed for the conversion of cellulose. In Chapter 5 we report the heterologous expression of endoglucanases and exoglucanases by G. thermodenitrificans T12, in an attempt to complement the enzymatic machinery of this strain and its suitability for consolidated bioprocessing. A metagenome screen was performed on the metagenome of 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-βglucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having most activity towards p-nitrophenyl-β-dxylopyranoside. Structure modelling of GE40 revealed a protein architecture similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic analysis against 2-HE-cellulose, CM-cellulose and barley β-glucan. In addition, we successfully expressed the earlier characterized Geobacillus sp. 70PC53 endoglucanase celA and the C. thermocellum exoglucanase celK in strain G. thermodenitrificans T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of Geobacillus thermodenitrificans T12 as a host for consolidated bioprocessing. In Chapter 6, we provided more insight in the genetic variation of the hemicellulolytic utilization cluster of G. thermodenitrificans. This variation is far greater than described before and gives ample opportunities for the further development of Geobacillus spp. for hemicellulose degradation. The production of cellulases in Geobacillus species is demonstrated to be successful, and we have expanded on that knowledge with the expression of both endo and exoglucanases from C. thermocellum. However, in line with previous studies, direct cellulose fermentation by geobacilli is not yet achieved, most likely due to insufficient cellulase production and/or secretion. With a rapidly expanding genetic toolbox for thermophiles, now including a thermostable Cas9, we expect that the successful development of Geobacillus spp. for consolidated bioprocessing is just a matter of time.</p

Rainforest-to-pasture conversion stimulates soil methanogenesis across the Brazilian Amazon

The Amazon rainforest is a biodiversity hotspot and large terrestrial carbon sink threatened by agricultural conversion. Rainforest-to-pasture conversion stimulates the release of methane, a potent greenhouse gas. The biotic methane cycle is driven by microorganisms; therefore, this study focused on active methane-cycling microorganisms and their functions across land-use types. We collected intact soil cores from three land use types (primary rainforest, pasture, and secondary rainforest) of two geographically distinct areas of the Brazilian Amazon (Santarém, Pará and Ariquemes, Rondônia) and performed DNA stable-isotope probing coupled with metagenomics to identify the active methanotrophs and methanogens. At both locations, we observed a significant change in the composition of the isotope-labeled methane-cycling microbial community across land use types, specifically an increase in the abundance and diversity of active methanogens in pastures. We conclude that a significant increase in the abundance and activity of methanogens in pasture soils could drive increased soil methane emissions. Furthermore, we found that secondary rainforests had decreased methanogenic activity similar to primary rainforests, and thus a potential to recover as methane sinks, making it conceivable for forest restoration to offset greenhouse gas emissions in the tropics. These findings are critical for informing land management practices and global tropical rainforest conservation

Exploiting the Anaerobic Expression of Pyruvate Dehydrogenase for the Production of Biofuels

The Pyruvate dehydrogense complex (PDH) is a primarily aerobic enzyme which catalyses pyruvate to acetyl-CoA and carbon dioxide. Its counterpart in anaerobic metabolism is pyruvate formate lyase (Pfl) which converts pyruvate to acetyl-CoA and formate. A novel fermentation pathway involving PDH rather than Pfl (or equivalent), which retains the reducing equivalents from pyruvate oxidation, could provide a novel route for ethanol production, as well as changes in redox balance opening up opportunities for the production of higher alcohols such as butanol. Utilising PDH for the production of biofuels has been investigated in three microorganisms: Geobacillus thermodenitrificans, Bacillus subtilis, and E. coli. Geobacillus thermodenitrificans does express Pfl, thus PDH is always active in the G. thermodenitrificans regardless of whether the bacterium is growing in aerobic or anaerobic conditions. To utilise this PDH in the production of ethanol a bi-functional alcohol dehydrogenase (AdhE) was introduced to G. thermodenitrificans K1041. Further optimisation of ethanol production was achieved by knocking-out lactate dehydrogenase (Ldh), which would otherwise compete with ethanol for flux from acetyl-CoA, and activity of the PDH promoter verses potential alternative promoters to increase the expression of the native PDH was investigated. Like G. thermodenitrificans, Bacillus subtilis also does not have a PFL pathway, but does have a native Adh so can undergo fermentation, albeit poorly. To increase ethanol production competing fermentation pathways were knocked-out, however this resulted in strains which were unable to grow anaerobically. The activity of the native PDH promoter was investigated, and PDH subsequently upregulated. The production of 1-butanol from B. subtilis was also achieved using expression of Clostridial genes encoding a butanol synthetic pathway from a plasmid and from chromosomal integrations. PDH in Gram-negative bacteria such as E. coli are not active during anerobic growth due to fermentation resulting in elevated levels of intracellular NADH; which in turn triggers negative feedback inhibition of PDH. A consequence of this is E. coli strains which are engineered to produce increased titres of ethanol by knocking-out pfl are unable to grow anaerobically. To alleviate this problem a PDH from gram-positive bacteria was expressed in E. coli. The effect of these PDH was also used to assess their potential benefits on 1-butanol in E. coli, by introducing Clostridial genes encoding a butanol synthetic pathway via plasmids

DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA

One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and bioremediation. For most applications, robust cellular systems are preferred for longer activity half-life and resistance to harsh environments. Two projects related to robust cellular systems involving Gram-positive bacteria are presented in this work. One is to develop thermostable genetic reporters for Geobacilli species and the other is to display an enzyme on the Bacillus subtilis spore surface to enhance its robustness and present an alternative to purified enzymes for industrial applications. Bacillus subtilis and Geobacillus thermoglucosidans are gram-positive, spore-forming bacteria. They secrete many proteins used industrially for the production of paper, food, textiles, chemicals, medicine, and cosmetics. Since G. thermoglucosidans is thermostable with an optimal growth temperature of 60ºC, its secreted proteins are also thermostable which proves advantageous for a variety of industrial applications. Additionally, a strain of G. thermoglucosidans has been used for the production of ethanol from biomass. Unfortunately the inner workings of G. thermoglucosidans are still poorly understood and a genetic toolkit is necessary to better discover how to improve them via genetic engineering for industrial use. Important components of this toolkit are genetic reporters which allow for the analysis of gene expression in G. thermoglucosidans. Fluorescent proteins are commonly used reporters for other bacterial species due to their easily observed and readily measured signal, however no thermostable fluorescent proteins have been shown to be functional in Geobacillus. Seven different fluorescent proteins including mCherry, Venus, GFP, sfGFP, GFPmut3, mCherry (Gt), and Venus (Gt) were tested for stability and functionality in Geobacillus thermoglucosidans. Venus (Gt) and mCherry (Gt) were codon optimized for this bacterium with the goal of increasing expression level and thus improving the fluorescence signal. The fluorescence intensity of each fluorescent protein expressed in G. thermoglucosidans was measured after several hours of bacterial growth at 50ºC and 60ºC. Venus, mCherry, Venus (Gt), mCherry (Gt), and sfGFP all had signal when expressed in G. thermoglucosidans at 50ºC and sfGFP had signal at 60ºC. Therefore, fluorescent reporter proteins in three different colors were found to be functional in G. thermoglucosidans. This will further genetic engineering of the species for thermostable protein production, bioremediation, and biofuel production. Bacillus subtilis is Generally Regarded as Safe (GRAS) by the FDA and amenable toward genetic manipulation. Thus it has been engineered for the production of many heterologous proteins. Oftentimes, proteins secreted by bacteria are purified for industrial use. However, protein purification is expensive and time-consuming and long-term storage of purified proteins requires extremely low temperatures (-20ºC). B. subtilis spores have been used to immobilize a variety of proteins for vaccines, biosensors, and bioremediation applications. Spore surface display eliminates the need for purification and provides a way to easily separate proteins from the final product if necessary. A novel and thermostable laccase, a copper-containing oxidase, was isolated and purified from G. thermoglucosidans. It can be used to degrade lignin and a variety of phenolic compounds and thus has applications for the production of paper, textiles, food, and biofuel. This laccase was isolated, characterized, and immobilized on the surface of B. subtilis spores. The purified and spore displayed laccase were tested for heat stability and catalytic function. The purified laccase showed high activity toward 2,6-dimethoxyphenol (2,6-DMP) and moderate activity toward veratryl alcohol and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) while the spore displayed laccase showed high activity toward 2,6-DMP. The purified laccase was considerably more heat stable than a commonly used fungal laccase. The spore displayed laccase was also found to be heat stable with a half-life of about 6 hours at 80ºC. The binding affinity of the immobilized laccase for the substrate 2,6-DMP was virtually the same as that of the purified laccase, plus the immobilized laccase showed solid activity. These results show that spore surface display of proteins is a promising, more inexpensive alternative to purifying proteins for industrial use

Microbial diversity gradients in the geothermal mud volcano underlying the hypersaline Urania Basin

Mud volcanoes transport deep fluidized sediment and their microbial communities and thus provide a window into the deep biosphere. However, mud volcanoes are commonly sampled at the surface and not probed at greater depths, with the consequence that their internal geochemistry and microbiology remain hidden from view. Urania Basin, a hypersaline seafloor basin in the Mediterranean, harbors a mud volcano that erupts fluidized mud into the brine. The vertical mud pipe was amenable to shipboard Niskin bottle and multicorer sampling and provided an opportunity to investigate the downward sequence of bacterial and archaeal communities of the Urania Basin brine, fluid mud layers and consolidated subsurface sediments using 16S rRNA gene sequencing. These microbial communities show characteristic, habitat-related trends as they change throughout the sample series, from extremely halophilic bacteria (KB1) and archaea (Halodesulfoarchaeum spp.) in the brine, toward moderately halophilic and thermophilic endospore-forming bacteria and uncultured archaeal lineages in the mud fluid, and finally ending in aromatics-oxidizing bacteria, uncultured spore formers, and heterotrophic subsurface archaea (Thermoplasmatales, Bathyarchaeota, and Lokiarcheota) in the deep subsurface sediment at the bottom of the mud volcano. Since these bacterial and archaeal lineages are mostly anaerobic heterotrophic fermenters, the microbial ecosystem in the brine and fluidized mud functions as a layered fermenter for the degradation of sedimentary biomass and hydrocarbons. By spreading spore-forming, thermophilic Firmicutes during eruptions, the Urania Basin mud volcano likely functions as a source of endospores that occur widely in cold seafloor sediments