Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Medium-chain esters are versatile chemicals with broad applications as flavors, fragrances, solvents, and potential drop-in biofuels. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either lipases or alcohol acyltransferase (AAT), but the AAT-dependent pathway is more thermodynamically favorable in an aqueous fermentation environment. Even though a cellulolytic thermophile Clostridium thermocellum harboring an AAT-dependent pathway has recently been engineered for direct conversion of lignocellulosic biomass into esters, the production is not efficient. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. The challenge is to identify and disrupt CEs that can alleviate ester degradation while not negatively affecting the efficient and robust capability of C. thermocellum for lignocellulosic biomass deconstruction. In this study, by using bioinformatics, comparative genomics, and enzymatic analysis to screen a library of CEs, we identified and disrupted the two most critical CEs, Clo1313_0613 and Clo1313_0693, that significantly contribute to isobutyl acetate degradation in C. thermocellum. We demonstrated that an engineered esterase-deficient C. thermocellum strain not only reduced ester hydrolysis but also improved isobutyl acetate production while maintaining effective cellulose assimilation.     

Interactions between the 2.4 and 4.2 regions of sigmaS, the stress-specific sigma factor of Escherichia coli, and the -10 and -35 promoter elements

The σ^s subunit of Escherichia coli RNA polymerase holoenzyme (Eσ^S) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by Eσ^S and the housekeeping Eσ⁷⁰ is as yet not clearly understood. We used a genetic approach to investigate the interaction of σ^S with its target promoters. Starting with down-promoter variants of a σ^S promoter target, osmEp, altered in the –10 or –35 elements, we isolated mutant forms of σ^S suppressing the promoter defects. The activity of these suppressors on variants of osmEp and ficp, another target of σ^S, indicated that σ^S is able to interact with the same key features within a promoter sequence as σ⁷⁰. Indeed, (i) σ^S can recognize the –35 element of some but not all its target promoters, through interactions with its 4.2 region; and (ii) amino acids within the 2.4 region participate in the recognition of the –10 element. More specifically, residues Q152 and E155 contribute to the strong preference of σ^S for a C in position –13 and residue R299 can interact with the –31 nucleotide in the –35 element of the target promoters.     

Whole-Genome Optical Mapping and Finished Genome Sequence of Sphingobacterium deserti sp. nov., a New Species Isolated from the Western Desert of China

A novel Gram-negative bacterium, designated ZW^T, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5–42°C with an optimum of 30°C, and at pH 7.0–11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c or C_16:1 ω6c/C_16:1 ω7c) (39.22%), iso-C15:0 (27.91%), iso-C_17:0 3OH (15.21%), C_16:0 (4.98%), iso-C_15:0 3OH (3.03%), C16:0 3OH (5.39%) and C_14:0 (1.74%). The major polar lipid of strain ZW^T is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZW^T represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZW^T (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZW^T, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZW^T features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZW^T with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZW^T to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.     

The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τ_C16. We show that the extreme C-terminal region of τ_C16 constitutes the site of interaction with α. The τ_C16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τ_C16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (K_D) of the α−τ_C16 complex to be ~260pM. Competition with immobilized τ_C16 by τ_C16 derivatives for binding to α gave values of K_D of 7μM for the α−τ_C16Δ7 complex. Low-level expression of the genes encoding τ_C16 and τ_C167, but not τ_C16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τ_C16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τ_C24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τ_C16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.     

Integrating Ontogenetic Shift,Growth and Mortality to Determine a Species' Ecological Role from Isotopic Signatures

Understanding species linkages and energy transfer is a basic goal underlying any attempt at ecosystem analysis. Although the first food-web studies were based on gut contents of captured specimens, the assessment of stable isotopes, mainly δ¹³C and δ¹⁵N, has become a standard methodology for wide-range analyses in the last 30 years. Stable isotopes provide information on the trophic level of species, food-web length, and origin of organic matter ingested by consumers. In this study, we analyzed the ontogenetic variability of δ¹³C and δ¹⁵N obtained from samples of three Neotropical fish species: silver sardine (Lycengraulis grossidens, n=46), white lambari (Cyanocharax alburnus, n= 26), and the red-tail lambari (Astyanax fasciatus, n=23) in Pinguela Lagoon, southern Brazil. We developed a new metric, called the Weighted Isotopic Signature (φ ¹⁵N or φ ¹³C, ‰), that incorporates ontogenetic variability, body growth, and natural mortality into a single number.