Pharmacokinetics of amikacin after intravenous, intra-articular, and combined intravenous and intra-articular administration in healthy neonatal foals.

BACKGROUND: Pharmacokinetics of amikacin administered IV to neonatal foals are described, but little data are available regarding the plasma concentrations contributed by concurrent intra-articular (IA) administration. HYPOTHESIS/OBJECTIVES: Compare the pharmacokinetics of amikacin when the total dose is administered IV compared to being divided between IV and IA routes of administration in neonatal foals and predict the plasma concentrations from various combined IV and IA dosing regimens. ANIMALS: Eight healthy neonatal foals. METHODS: Foals received 3 amikacin treatment protocols: (1) IV-only (25 mg/kg q24h IV), (2) concurrent IV and IA (16.7 mg/kg q24h IV and 8.3 mg/kg q24h into 1 tarsocrural joint), and (3) IA-only (8.3 mg/kg q24h into 1 tarsocrural joint). Protocols were administered for 3 days beginning at 7, 14, and 21 days of age. Plasma concentrations ≥53 µg/mL at 30 minutes were considered therapeutic for isolates with intermediate susceptibility. RESULTS: Foal age was a significant variable. The IV-only protocol met or exceeded the 30-minute plasma concentrations considered therapeutic (mean µg/mL [95% confidence interval, CI]) in 7- to 9-day-old (54.0 [52.2-56.9]), 14- to 16-day-old (58.1 [55.2-61.0]), and 21- to 23-day-old (66.6 [63.7-69.6]) foals. Concurrent IV and IA protocol did not reach the 30-minute concentration considered therapeutic in 7- to 9-day-old foals (46.5 [43.6-49.4]) but did in 14- to 16-day-old (62.9 [60.0-65.8]) and 21-to 23-day-old (62.6 [59.7-65.6]) foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Concurrent IV and IA administration of amikacin produces 30-minute plasma concentrations considered therapeutic in foals 14 to 23 days old, but concentrations observed in younger foals might be below those considered therapeutic for isolates with intermediate susceptibility to amikacin.

Partial ceratohyoidectomy as surgical treatment for horses with temporohyoid osteoarthropathy: 10 cases (2010-2021).

OBJECTIVE: To describe outcomes of horses with temporohyoid osteoarthropathy (THO) treated with partial ceratohyoidectomy. ANIMALS: 10 client-owned horses. PROCEDURES: Medical records from 2 institutions were examined for records of horses with THO treated with partial ceratohyoidectomy between 2010 and 2021. History, signalment, clinical signs, diagnostics, medications, and surgery-related details were recorded. Horses with a minimum of 6 months follow-up were recruited for neurologic and imaging examinations in the hospital or field where radiography of the basihyoid-ceratohyoid articulation were performed along with CT, when available. RESULTS: 10 horses with THO were included (9 unilateral; 1 bilateral). Nine planned partial ceratohyoidectomies were performed in 8 horses, whereas 2 horses had preoperatively planned complete ceratohyoidectomies transitioned to partial ceratohyoidectomies during surgery due to intraoperative complications. Postoperative complications occurred mostly in transitioned surgeries (obstructed airway, tongue mobility issues, and incisional hemorrhage), whereas only 1 horse with a planned ceratohyoidectomy had postoperative complication of rhabdomyolysis. All complications resolved before hospital discharge. Neurologic signs improved in all 10 horses, with 2 showing complete resolution. Nine horses were available for radiographic follow-up, 6 of which also had head CT scans. A space between the ceratohyoid and basihyoid bones was measurable on radiography in all 9 horses, and was confirmed on CT. Three horses demonstrated proliferation of either ceratohyoid or basihyoid bones. The 9 horses with unilateral disease returned to previous work, and the horse with bilateral disease was retired. CLINICAL RELEVANCE: Partial ceratohyoidectomy is a surgical option for treatment of THO that provides similar clinical outcomes to published reports on ceratohyoidectomy.

Navicular bone fracture and severe deep digital flexor tendinopathy after palmar digital neurectomy in two horses.

Background: Navicular syndrome is a common cause of forelimb lameness in horses. Beyond changes to the navicular bone itself, horses with a clinical diagnosis of navicular syndrome often have pathology associated with other components of the navicular apparatus, including the navicular bursa, deep digital flexor (DDF) tendon, collateral sesamoidean ligaments, and impar ligament. Palmar digital neurectomy (PDN) is often used as a salvage procedure for horses diagnosed with navicular syndrome that become unresponsive to medical management. There are many potential complications associated with PDN, some of which are debilitating. Case Description: This report describes two cases of navicular bone fracture with severe DDF tendinopathy and distal interphalangeal joint subluxation/hyperextension that occurred 12 and 19 weeks after bilateral forelimb PDN. Conclusion: These two cases highlight the importance of proper patient selection before PDN due to the high incidence of undiagnosed soft tissue pathology in conjunction with radiographic evidence of navicular syndrome. Advanced imaging of the digit is recommended to identify and characterize any soft tissue pathology associated with the navicular apparatus before pursuing PDN to avoid disease progression and catastrophic injury.

Adaptive Synthesis of a Rough Lipopolysaccharide in Geobacter sulfurreducens for Metal Reduction and Detoxification.

The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor fumarate but expressed only the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (ΔrfaQ) or complete (ΔrfaC) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in surface charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for extracellular mineralization of the toxic uranyl cation but were unable to prevent permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal-rich environments and their performance in bioremediation and bioenergy applications. IMPORTANCE Some metal-reducing bacteria produce an LPS without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature was not previously investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.

Efficacy of orally administered gabapentin in horses with chronic thoracic limb lameness.

OBJECTIVE: To evaluate the analgesic effects of orally administered gabapentin on horses with chronic thoracic limb lameness. STUDY DESIGN: Randomized, crossover design. ANIMALS: A total of 14 adult horses with chronic thoracic limb lameness. METHODS: Following baseline measurement of lameness, horses were administered each of four treatments orally in grain: treatment G, gabapentin (20 mg kg-1) twice daily for 13 doses; treatment F, firocoxib (171 mg once, then 57 mg once daily for six doses); treatment GF, gabapentin and firocoxib at previously stated doses and frequencies; or treatment C, grain only as a control. Treatments were administered in a randomized, crossover design, separated by 2 weeks. Subjective lameness score (SLS), inertial sensor vector sum (VS) calculations, peak vertical ground reaction force (PVGRF) measurements and vertical impulse (VI) calculations were determined immediately prior to each initial treatment dose and 2-4 hours after the final treatment dose for each treatment. Mean change in SLS, VS, PVGRF and VI for each treatment were compared among treatments. RESULTS: The rank change in SLS of treatment GF was significantly greater than that of treatments C (p = 0.01) and G (p = 0.01) but not of treatment F (p = 0.08). No differences in VS (p = 0.4), PVGRF (p = 0.4) or VI (p = 0.1) were observed among treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Gabapentin, as administered here, did not improve subjective or objective measures of lameness in horses with chronic thoracic limb musculoskeletal pain. Although subjective evaluation identified an improvement in lameness with treatment GF, it was not different from that observed with treatment F. Higher oral dosing and longer treatment regimens of gabapentin may be indicated for the treatment of chronic musculoskeletal pain in horses.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content.

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

Subtourniquet pressures generated by application of wide-rubber tourniquets in standing, sedated horses.

OBJECTIVE: To determine the influence of location and movement on subtourniquet pressure (STP) generated by application of a wide-rubber tourniquet (WRT) on equine limbs. STUDY DESIGN: Randomized experimental cross-over design. ANIMALS: Six standing, sedated horses. METHODS: Horses were sedated with detomidine hydrochloride (0.2 µg/kg IV), and 4 investigators applied WRTs to the antebrachium (AB), gaskin (GK), and midmetacarpus (MC) of each horse in a predetermined, randomized order. Subtourniquet pressure was consequently measured at 10-minute intervals (T0, T10, T20, T30) for 30 minutes. Indirect systolic blood pressure (SBP) was measured presedation, postsedation, and throughout the tourniquet application period. Target STP was established as SBP + 100 mm Hg. Limb movements at each location were classified as none, low, moderate, or high, on the basis of counts and magnitude. RESULTS: Mean STP did not change with time (P = .93) and exceeded SBP by 163 mm Hg (95% CI 122-205), 185 mm Hg (95% CI 156-214), and 402 mm Hg (95% CI 351-454) at the AB, GK, and MC, respectively. Mean STP at each location exceeded the target STP in 59 of 70 (81%) of the trials. Limb movements affected STP generated by tourniquets at the AB (P = .04) and MC (P < .0001) but not at the GK (P = .67). CONCLUSION: Wide-rubber tourniquets applied at the AB, GK, and MC generated STP >100 mm Hg above SBP for 30 minutes in standing, sedated horses. CLINICAL SIGNIFICANCE: Wide-rubber tourniquets as applied in this study can achieve and maintain the current recommended STP (SBP + 100 mm Hg) for equine IV regional limb perfusion. Number and magnitude of limb movement can decrease STP over time, potentially reducing the efficacy of a WRT.

Quantitative assessment of intravenous regional limb perfusion of tiludronate as an adjunctive treatment for lameness caused by navicular syndrome in horses.

OBJECTIVE To determine effects for 2 IV regional limb perfusion (IVRLP) protocols involving tiludronate on lameness of horses with navicular syndrome. ANIMALS 15 horses with bilateral forelimb navicular syndrome. PROCEDURES Shoeing and anti-inflammatory injection into the distal interphalangeal joint (DIPJ) of both forelimbs (day 0) were performed on all horses. On day 14, horses received 1 of 3 IVRLPs consisting of 0.1 mg of tiludronate/kg (low-dose tiludronate [LDT]; n = 5), 0.2 mg of tiludronate/kg (high-dose tiludronate [HDT]; 5), or saline (0.9% NaCl) solution (placebo; 5); treatments were repeated at days 24 and 34. Lameness severity of both forelimbs was evaluated via subjective evaluation and force plate analysis before and after shoeing on day 0 and at days 14, 34, 60, and 120. Mean subjective lameness score and peak vertical ground reaction force (PVGRF) for the more severely lame forelimb (LFL) and both (combined) forelimbs (CFL) were compared over time. RESULTS For all horses, mean PVGRF for the LFL and CFL was increased at 14 days. No difference in mean subjective lameness score or mean PVGRF was detected within groups at any time. Mean PVGRF of the CFL was higher for the HDT group than the LDT and placebo groups only at 120 days. CONCLUSIONS AND CLINICAL RELEVANCE Use of the tiludronate IVRLP protocols described here provided no further improvement in lameness over therapeutic shoeing and anti-inflammatory injection of the DIPJ in horses with navicular syndrome. However, HDT-treated horses were objectively less lame than LDT- or placebo-treated horses at 120 days.

High activity CAZyme cassette for improving biomass degradation in thermophiles.

BACKGROUND: Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. RESULTS: To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even  improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. CONCLUSIONS: We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

Effects of tourniquet number and exsanguination on amikacin concentrations in the radiocarpal and distal interphalangeal joints after low volume intravenous regional limb perfusion in horses.

OBJECTIVE: To determine the influence of a dual tourniquet technique and limb exsanguination on amikacin concentrations in the synovial fluid of the radiocarpal joint (RCJ) and distal interphalangeal joint (DIPJ) after low volume, cephalic intravenous regional limb perfusion (IVRLP). STUDY DESIGN: Randomized cross-over design. ANIMALS: Six healthy adult horses. METHODS: One gram of amikacin in 6 mL of 0.9% NaCl was infused via cephalic IVRLP in 6 standing, sedated horses using 4 techniques: proximal pneumatic tourniquet (P), proximal pneumatic tourniquet with exsanguination (PE), proximal pneumatic and distal Esmarch tourniquet (PD), and proximal pneumatic with distal Esmarch tourniquet and exsanguination (PDE). Amikacin concentrations were measured in RCJ and DIPJ synovial fluid samples, collected just before perfusion (time 0), and at 15 and 30 minutes (before tourniquet release) after perfusion. RESULTS: Synovial fluid amikacin concentrations achieved in the RCJ were higher with techniques PD and PDE than those achieved with techniques P and PE 15 and 30 minutes after perfusion (P < .0001). Synovial fluid amikacin concentrations in the DIPJ were higher with techniques P and PE than those achieved with techniques PD and PDE at 15 minutes (P = .0002) and were higher than technique PDE at 30 minutes after perfusion (P < .0001). CONCLUSION: Low volume (10 mL) cephalic IVRLP should be combined with the placement of 2 tourniquets (proximal and distal to the carpus) to achieve therapeutic amikacin concentrations in the RCJ. Exsanguination prior to low volume IVRLP does not alter synovial fluid amikacin concentrations.

Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose.

BACKGROUND: The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a ß-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-ß-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. We tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity. RESULTS: In vitro analysis of E1 and CelA shows synergistic interaction. The E1 gene from Acidothermus cellulolyticus was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. The vector was integrated into the C. bescii chromosome at a site previously showing no detectable detrimental consequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C. CONCLUSIONS: Expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome. These data agree with in vitro evidence that E1 acts synergistically with CelA to digest cellulose and offer the possibility of engineering additional enzymes for improved biomass deconstruction with the knowledge that C. bescii can express a gene from Acidothermus, and perhaps other heterologous genes, effectively.

Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.

Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass.

BACKGROUND: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii, which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities. RESULTS: A deletion of the celA gene resulted in a dramatic reduction in the microorganism's ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains. CONCLUSIONS: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction.

BACKGROUND: Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria. RESULTS: To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host. CONCLUSIONS: The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.

Fermentation of glycerol into ethanol in a microbial electrolysis cell driven by a customized consortium.

The in situ generation of ethanol from glycerol-containing wastewater shows promise to improve the economics of the biodiesel industry. Consequently, we developed a microbial electrolysis cell (MEC) driven by the synergistic metabolisms of the exoelectrogen Geobacter sulfurreducens and the bacterium Clostridium cellobioparum, which fermented glycerol into ethanol in high yields (90%) and produced fermentative byproducts that served as electron donors for G. sulfurreducens. Syntrophic cooperation stimulated glycerol consumption, ethanol production, and the conversion of fermentation byproducts into cathodic H2 in the MEC. The platform was further improved by adaptively evolving glycerol-tolerant strains with robust growth at glycerol loadings typical of biodiesel wastewater and by increasing the buffering capacity of the anode medium. This resulted in additional increases in glycerol consumption (up to 50 g/L) and ethanol production (up to 10 g/L) at rates that greatly exceeded the capacity of the anode biofilms to concomitantly remove the fermentation byproducts. As a result, 1,3-propanediol was generated as a metabolic sink for electrons not converted into electricity syntrophically. The results highlight the potential of consortia to process glycerol in MECs and provide insights into genetic engineering and system design approaches that can be implemented to further improve MEC performance to satisfy industrial needs.

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability.

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.