A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

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Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii. Furthermore, a facile approach for assessing genomic stability in C. bescii has been established.IMPORTANCE Caldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

Williams-Rhaesa

Poole

Dinsmore

et al. 2017

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“…Cultures of strain UFO1 (50 ml) were grown to late log phase (20 h) in modified R2 broth without (in triplicate experiments) and with (in duplicate experiments) 50 M uranium added to the growth medium. RNA was isolated from the cultures and reverse transcription-PCR (RT-PCR) was performed as previously described (32). Primers for RT-PCR are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

Thorgersen

Lancaster

Rajeev

et al. 2017

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Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound.IMPORTANCE Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments.KEYWORDS contamination, uranium sequestration, uranium-binding protein U ranium is highly toxic and is also a radionuclide with a long half-life (1). Uranium mining, milling, and other anthropogenic industrial activities have led to widespread environmental contamination with associated hazards to human health and to the environment (2). In the United States, the U.S. Department of Energy (DOE) oversees the monitoring and restoration of waste sites at 12 facilities contaminated with uranium (3). Uranium is present in oxic fresh water as highly soluble U(VI). At pH values lower than 5.0, U(VI) is predominantly found as the free uranyl ion (UO 2 2ϩ ), and between pH values of 5.0 and 7.5, U(VI) is predominantly complexed with carbonate. At pH values

“…For example, it has been used in developing a replicating shuttle vector for C. bescii and other Caldicellulosiruptor species (9), and this vector has been used for homologous overexpression of the genes celA, encoding a bifunctional glycoside hydrolase (10), and xor, encoding a tungsten-dependent oxidoreductase (11). Numerous gene deletions in addition to cbeI have also been constructed, including the ldh gene encoding lactate dehydrogenase (12), a gene cluster encoding pectin-degrading enzymes (13), the celA gene (14), and a gene cluster encoding [Ni-Fe] hydrogenase maturation enzymes (15).…”

mentioning

confidence: 99%

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

Lipscomb

Conway

Blumer-Schuette

et al. 2016

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Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii. These strains showed resistance to kanamycin at concentrations >50 g · ml ؊1 , whereas wild-type C. bescii was sensitive to kanamycin at 10 g · ml ؊1 . In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ⌬pyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCECaldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism. Caldicellulosiruptor bescii is a thermophilic anaerobic Grampositive bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass (1, 2). It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel product...