Characterization of the plasmids harbouring the florfenicol resistance gene floR in Glaesserella parasuis and Actinobacillus indolicus.

OBJECTIVES: The aim of this study was to characterize the floR-carrying plasmids originating from Glaesserella parasuis and Actinobacillus indolicus isolated from pigs with respiratory disease in China. METHODS: A total of 125 G. parasuis and 28 A. indolicus strains collected between 2009 and 2022 were screened for florfenicol resistance. Characterization of floR-positive isolates and plasmids were determined by antimicrobial susceptibility testing, serotyping, multilocus sequence typing (MLST), conjugation and transformation assays, whole-genome sequencing (WGS), and phylogenetic analysis. RESULTS: One A. indolicus and six G. parasuis were identified as positive for floR. The six G. parasuis were assigned to four different serovars, including serovars 6, 7, 9, and unknown. In addition to strain XP11, six floR genes were located on plasmids. The six floR-bearing plasmids could be transformed into Pasteurella multocida and divided into two different types, including ∼5000 bp and ∼6000 bp plasmids. The ∼5000 bp plasmids consisting of rep, lysR, mobB, and floR genes, exhibited high similarity among Pasteurellaceae bacteria. Furthermore, the ∼6000 bp plasmids, consisting of rep, lysR, mobC, mobA/L, and floR genes, showed high similarity between G. parasuis and Actinobacillus Spp. Notably, WGS results showed that the floR modules of the two types of plasmids could be transferred and integrated into the diverse Pasteurellaceae- origined plasmids. CONCLUSION: This study firstly reported the characterization of floR-carrying plasmids from A. indolicus and a non-virulent serovar of G. parasuis in pigs in China and elucidated the transmission mechanism of the floR resistance gene among the Pasteurellaceae family.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates.

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Transcriptional regulation in key metabolic pathways of Actinobacillus succinogenes in the presence of electricity.

The potential of renewable energy application via direct electrode interaction for the production of bio-based chemicals is a promising technology. The utilization of extracellular energy in pure culture fermentations aims in intracellular redox balance regulation in order to improve fermentation efficiency. This work evaluates the impact of a bioelectrochemical system in succinic acid fermentation and the metabolic response of Actinobacillus succinogenes. The metabolic pathway regulation of A. succinogenes was evaluated via RNA expression of the key enzymes that participate in TCA cycle, pyruvate metabolism and oxidative phosphorylation. The genes that were significantly overexpressed in BES compared to non-BES were phosphoenolpyruvate carboxykinase (0.4-fold change), inorganic pyrophosphatase (2.3-fold change) and hydrogenase (2.2-fold change) and the genes that were significantly underexpressed were fumarase (-0.94-fold change), pyruvate kinase (-6.9-fold change), all subunits of fumarate reductase (-2.1 to -1.17-fold change), cytochromes I and II (-1.25 and -1.02-fold change, respectively) and two C4-carboxylic acid transporters.

One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z.

Within the field of bioproduction, non-model organisms offer promise as bio-platform candidates. Non-model organisms can possess natural abilities to consume complex feedstocks, produce industrially useful chemicals, and withstand extreme environments that can be ideal for product extraction. However, non-model organisms also come with unique challenges due to lack of characterization. As a consequence, developing synthetic biology tools, predicting growth behavior, and building computational models can be difficult. There have been many advancements that have improved work with non-model organisms to address broad limitations, however each organism can come with unique surprises. Here we share our work in the non-model bacterium Actinobacillus succinognes 130Z, which includes both advancements in synthetic biology toolkit development and pitfalls in unpredictable fermentation behaviors. To develop a synthetic biology "tool kit" for A. succinogenes, information gleaned from a growth study and antibiotic screening was used to characterize 22 promoters which demonstrated a 260-fold range of fluorescence protein expression. The strongest of the promoters was incorporated into an inducible system for tunable gene control in A. succinogenes using the promoter for the lac operon as a template. This system flaunted a 481-fold range of expression and no significant basal expression. These findings were accompanied by unexpected changes in fermentation products characterized by a loss of succinic acid and increase in lactic acid after approximately 10 months in the lab. During evaluation of the fermentation shifts, new tests of the synthetic biology tools in a succinic acid producing strain revealed a significant loss in their functionality. Contamination and mutation were ruled out as causes and further testing is needed to elucidate the driving factors. The significance of this work is to share a successful tool development strategy that could be employed in other non-model species, report on an unfortunate phenomenon that needs addressed for further development of A. succinogenes, and provide a cautionary tale for those undertaking non-model research. In sharing our findings, we seek to provide tools and necessary information for further development of A. succinogenes as a platform for bioproduction of succinic acid and to illustrate the importance of diligent and long-term observation when working with non-model bacteria.

Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin.

The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.

Succinic acid production from pulp and paper industry waste: A transcriptomic approach.

The fermentative production of biobased chemicals and polymers using crude lignocellulose hydrolysates is challenging due to the presence of various inhibitory compounds and multiple sugars. This study evaluates the metabolic response of Actinobacillus succinogenes for the production of succinic acid using spent sulphite liquor (SSL) as feedstock derived from industrial acidic sulphite pulping of Eucalyptus globulus hardwood. A transcriptomic approach led to significant insights on gene regulation of the major metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle, pyruvate metabolism and oxidative phosphorylation) in batch cultures carried out on SSL and compared with glucose and xylose. Significantly overexpressed genes in SSL compared to glucose and xylose were fructose biphosphate aldolase (> 1.18-fold change) in the catabolism, phosphoenolpyruvate carboxykinase (> 1.59-fold change) and malate dehydrogenase (> 1.49-fold change) in the TCA cycle, citrate lyase (> 1.7-fold change), dihydrolipoamide dehydrogenase (> 0.88-fold change), pyruvate dehydrogenase E2 (> 1.63-fold change) and pyruvate formate lyase (> 0.61-fold change), involved in acetyl-CoA pathways. Finally, C4 tricarboxylic transporters were overexpressed (DCU (> 1.61-fold change) and 0079 (> 4.19-fold change). SSL was responsible for the upregulation of genes involved in the TCA cycle and oxidative phosphorylation, while xylose showed similar results with SSL in the oxidative phosphorylation.

Diverse strains of Actinobacillus lignieresii isolated from clinically affected cattle in a geographically restricted area.

OBJECTIVE: To investigate whether an outbreak of Actinobacillus lignieresii was caused by one or multiple strains. METHODS: Nine isolates of A. lignieresii were obtained from the lymph nodes of 15 affected cattle from two farms to determine whether a single strain was involved. An enterobacterial repetitive insertion consensus sequence (ERIC) PCR was used for genotyping, and the repeats-in-toxin genes were analysed by PCR and sequencing. RESULTS: Isolates from the two farms belonged to two and three genotypes, with a total of four genotypes detected. Genes of the apxICABD operons of some strains had deletions in the apxIA (~697 bp) and in the apxID (~187 bp) genes. The toxin gene deletions and the ERIC PCR patterns suggested the involvement of different A. lignieresii genotypes. CONCLUSION: There was no evidence that a unique genotype was associated with actinobacillosis on the two farms, confirming that this disease was associated with other contributing factors.

Actinobacillus species isolated from Japanese Thoroughbred racehorses in the last two decades.

Actinobacillus species are known to be pathogenic to horses. To clarify etiological agents of actinobacillosis in Japanese adult horses, 27 isolates from Japanese Thoroughbred racehorses putatively identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as Actinobacillus were further identified by PCR of the A. equuli toxin gene, by CAMP test, and by 16S rRNA sequencing analysis. Actinobacillus equuli subsp. haemolyticus was isolated most frequently (16/27) and was related to respiratory infections. Actinobacillus equuli subsp. equuli (4/27) was isolated from chronic cases or concomitant with other bacterial infections. The remainder were A. pleuropneumoniae, unclassified Actinobacillus species and Pasteurella caballi. Actinobacillus equuli including subsp. haemolyticus and subsp. equuli were the species most frequently isolated from equine actinobacillosis in Japan.

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

Investigation of putative invasion determinants of Actinobacillus species using comparative genomics.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

High-affinity l-malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4-dicarboxylates.

Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C4 -dicarboxylates requires transporters catalysing uptake and efflux of C4 -dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l-malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l-malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time-dependent uptake and competitive inhibition assays demonstrated that l-malate is the most preferred substrate for uptake by DcuE. The Vmax of DcuE for l-malate was 20.04 µmol/gDW·min with Km of 57 µM. The Vmax for l-malate was comparable to that for fumarate, whereas the Km for l-malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l-malate was 7.3-fold higher than that for fumarate, showing high efficiency and high affinity for l-malate. Furthermore, DcuE catalysed the reversible exchange of three C4 -dicarboxylates - l-malate, fumarate and succinate - but the preferred substrate for uptake was l-malate. Under physiological conditions, the C4 -dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l-malate/succinate antiporter in A. succinogenes.

Use of next generation sequencing to investigate the microbiota of experimentally induced wounds and the effect of bandaging in horses.

OBJECTIVES: To use next generation sequencing to characterize the microbiota of horses during healing of skin wounds in two anatomical locations (body and limb) known to present different healing patterns; and to investigate the impact of bandaging on bacterial communities of skin wounds located on the limbs of horses. METHODS: Full-thickness skin wounds were created on the distal extremity of both thoracic limbs and on one lateral mid-thoracic wall of four healthy horses. Limb wounds were randomly assigned to bandaging or not. A full-thickness sample was collected with a biopsy punch from intact thorax and limb skin (T0) and from the margin of one wound per site (thorax, unbandaged limb, bandaged limb) 1 week (T1) and 2 weeks (T2) postoperatively, and at full healing (T3). Thoracic skin samples obtained from three healthy horses were included in the analysis as controls. RESULTS: Anatomic location (thorax vs. limb) significantly influenced bacterial composition of equine skin and healing wounds. Fusobacterium and Actinobacillus were strongly associated with limb wounds during the initial phases of healing. Bandaging had a significant impact on the microbiota during the healing process. The skin microbiota after healing was more similar to samples from controls, demonstrating the resilience and stability of the environment. CONCLUSIONS: Equine skin microbiota is a rich and stable environment that is disturbed by wounding, but returns to its previous stage after full healing. Anatomic location significantly influences bacterial composition of the equine skin during wound healing. Bandaging has a significant impact on the skin microbiota of horses during the healing process. Results of this study provide new insight for a better understanding of the contribution of bacteria to wound healing in horses and may facilitate the future development of therapeutic strategies using commensal bacteria.

Reconstruction of a genome-scale metabolic model for Actinobacillus succinogenes 130Z.

BACKGROUND: Actinobacillus succinogenes is a promising bacterial catalyst for the bioproduction of succinic acid from low-cost raw materials. In this work, a genome-scale metabolic model was reconstructed and used to assess the metabolic capabilities of this microorganism under producing conditions. RESULTS: The model, iBP722, was reconstructed based on the functional reannotation of the complete genome sequence of A. succinogenes 130Z and manual inspection of metabolic pathways, covering 1072 enzymatic reactions associated with 722 metabolic genes that involve 713 metabolites. The highly curated model was effective in capturing the growth of A. succinogenes on various carbon sources, as well as the SA production under various growth conditions with fair agreement between experimental and predicted data. Calculated flux distributions under different conditions show that a number of metabolic pathways are affected by the activity of some metabolic enzymes at key nodes in metabolism, including the transport mechanism of carbon sources and the ability to fix carbon dioxide. CONCLUSIONS: The established genome-scale metabolic model can be used for model-driven strain design and medium alteration to improve succinic acid yields.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Transcriptome analysis and anaerobic C4 -dicarboxylate transport in Actinobacillus succinogenes.

A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C4 -dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270-0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [14 C]fumarate and [14 C]succinate were performed to experimentally verify that A. succinogenes possesses multiple C4 -dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5 mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C4 -dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (∆1999) decreased fumarate uptake significantly at approximately 5 mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 µmol·gDW-1 ·min-1 . These results suggest that C4 -dicarboxylate transport in A. succinogenes is mediated by multiple transporters, which transport various types and concentrations of C4 -dicarboxylates.

Natural lymphatic ("atypical") actinobacillosis in cattle caused by Actinobacillus lignieresii.

Bovine actinobacillosis is typically characterized by pyogranulomatous glossitis (wooden tongue). The involvement of other tissues, generally the skin or lymph nodes, has been regarded as atypical or cutaneous. We describe herein 2 outbreaks of actinobacillosis affecting primarily the lymph nodes of the head and neck. The disease affected 40 of 540 lactating cows in a dairy herd, and 5 of 335 two-y-old steers in a beef herd. Multiple or single, occasionally ulcerated nodules were observed in the region of the mandible, neck, and shoulder, including the parotid, submandibular, retropharyngeal, and prescapular lymph nodes. The histologic lesions were multifocal pyogranulomatous lymphadenitis, dermatitis, and cellulitis with Splendore-Hoeppli material. One steer had an exophytic pyogranuloma in the gingiva and another died because of ruminal tympany secondary to oropharyngeal and esophageal obstruction by a pyogranulomatous mass. Actinobacillus lignieresii was isolated from the lesions and identified by amplification, sequencing, and analysis of the 16S ribosomal (r)DNA gene. Seven of 8 cows recovered after treatment with sodium iodide. Lymphatic actinobacillosis is a frequent disease in Uruguay, southern Brazil, and Argentina. Morbidity is 1-50%; mortality is <1%. A. lignieresii apparently penetrates the intact oral and pharyngeal mucosa, infecting primarily the regional lymph nodes. Later, lesions may extend to the subcutaneous tissue and the skin, causing ulceration. Affected cattle with draining pyogranulomas contaminate the environment, favoring disease transmission, and should be treated with sodium iodide or antibiotics and isolated from the herd in order to control the disease.

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.

Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.

Production of homogeneous glycoprotein with multisite modifications by an engineered N-glycosyltransferase mutant.

Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma.

BACKGROUND: Oral mucositis is probably the most debilitating complication that can arise in treating a patient with head and neck cancer. Little is known about the impacts of oral microbiota on the initiation and progression of mucositis. METHODS: Based on 16S rRNA gene sequencing, dynamic changes in oral bacterial profile as well as correlations between the severity of mucositis and bacterial shifts during radiotherapy were investigated. FINDINGS: Our results revealed that bacterial community structure altered progressively during radiation therapy, in parallel with a marked increase in the relative abundance of some Gram-negative bacteria. Patients who eventually developed severe mucositis harbored a significantly lower bacterial alpha diversity and higher abundance of Actinobacillus during the phase of erythema - patchy mucositis. Accordingly, a random forest model for predicting exacerbation of mucositis was generated, which achieved a high predictive accuracy (AUC) of 0.89. INTERPRETATION: Oral microbiota changes correlate with the progression and aggravation of radiotherapy-induced mucositis in patients with nasopharyngeal carcinoma. Microbiota-based strategies can be used for the early prediction and prevention of the incidence of severe mucositis during radiotherapy.

Development of reactor configurations for an electrofuels platform utilizing genetically modified iron oxidizing bacteria for the reduction of CO2 to biochemicals.

Electrofuels processes are potentially promising platforms for biochemical production from CO2 using renewable energy. When coupled to solar panels, this approach could avoid the inefficiencies of photosynthesis and there is no competition with food agriculture. In addition, these systems could potentially be used to store intermittent or stranded electricity generated from other renewable sources. Here we develop reactor configurations for continuous electrofuels processes to convert electricity and CO2 to isobutyric acid (IBA) using genetically modified (GM) chemolithoautotrophic Acidithiobacillus ferrooxidans. These cells oxidize ferrous iron which can be electrochemically reduced. During two weeks of cultivation on ferrous iron, stable cell growth and continuous IBA production from CO2 were achieved in a process where media was circulated between electrochemical and biochemical rectors. An alternative process with an additional electrochemical cell for accelerated ferrous production was developed, and this system achieved an almost three-fold increase in steady state cell densities, and an almost 4-fold increase in the ferrous iron oxidation rate. Combined, this led to an almost 8-fold increase in the steady state volumetric productivity of IBA up to 0.063±0.012mg/L/h, without a decline in energy efficiency from previous work. Continued development of reactor configurations which can increase the delivery of energy to the genetically modified cells will be required to increase product titers and volumetric productivities.