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.

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.

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.

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.

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.

High-yield anaerobic succinate production by strategically regulating multiple metabolic pathways based on stoichiometric maximum in Escherichia coli.

BACKGROUND: Succinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli. RESULTS: In the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose). CONCLUSIONS: It's the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.

Sampling with poling-based flux balance analysis: optimal versus sub-optimal flux space analysis of Actinobacillus succinogenes.

BACKGROUND: Flux balance analysis is traditionally implemented to identify the maximum theoretical flux for some specified reaction and a single distribution of flux values for all the reactions present which achieve this maximum value. However it is well known that the uncertainty in reaction networks due to branches, cycles and experimental errors results in a large number of combinations of internal reaction fluxes which can achieve the same optimal flux value. RESULTS: In this work, we have modified the applied linear objective of flux balance analysis to include a poling penalty function, which pushes each new set of reaction fluxes away from previous solutions generated. Repeated poling-based flux balance analysis generates a sample of different solutions (a characteristic set), which represents all the possible functionality of the reaction network. Compared to existing sampling methods, for the purpose of generating a relatively "small" characteristic set, our new method is shown to obtain a higher coverage than competing methods under most conditions. The influence of the linear objective function on the sampling (the linear bias) constrains optimisation results to a subspace of optimal solutions all producing the same maximal fluxes. Visualisation of reaction fluxes plotted against each other in 2 dimensions with and without the linear bias indicates the existence of correlations between fluxes. This method of sampling is applied to the organism Actinobacillus succinogenes for the production of succinic acid from glycerol. CONCLUSIONS: A new method of sampling for the generation of different flux distributions (sets of individual fluxes satisfying constraints on the steady-state mass balances of intermediates) has been developed using a relatively simple modification of flux balance analysis to include a poling penalty function inside the resulting optimisation objective function. This new methodology can achieve a high coverage of the possible flux space and can be used with and without linear bias to show optimal versus sub-optimal solution spaces. Basic analysis of the Actinobacillus succinogenes system using sampling shows that in order to achieve the maximal succinic acid production CO2 must be taken into the system. Solutions involving release of CO2 all give sub-optimal succinic acid production.

Effects of eliminating pyruvate node pathways and of coexpression of heterogeneous carboxylation enzymes on succinate production by Enterobacter aerogenes.

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.

Effects of heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase on organic acid production in Aspergillus carbonarius.

Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates.

Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na(+). The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na(+)-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A. succinogenes).

Development of a markerless knockout method for Actinobacillus succinogenes.

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, ß-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

BACKGROUND: Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. RESULTS: The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. CONCLUSIONS: The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Enhanced succinic acid production by Actinobacillus succinogenes after genome shuffling.

Succinic acid is an important platform chemical for synthesis of C4 compounds. We applied genome shuffling to improve fermentative production of succinic acid by A. succinogenes. Using a screening strategy composed of selection in fermentation broth, cultured in 96-deep-well plates, and condensed HPLC screening, a starting population of 11 mutants producing a higher succinic acid concentration was selected and subjected to recursive protoplasts fusion. After three rounds of genome shuffling, strain F3-II-3-F was obtained, producing succinic acid at 1.99 g/l/h with a yield of 95.6 g/l. The genome shuffled strain had about a 73 % improvement in succinic acid production compared to the parent strain after 48 h in fed-batch fermentation. The genomic variability of F3-II-3-F was confirmed by amplified fragment-length polymorphism. The activity levels of key enzymes involved in end-product formation from glucose and metabolic flux distribution during succinic acid production were compared between A. succinogenes CGMCC 1593 and F3-II-3-F. Increased activity of glucokinase, fructose-1,6-bisphosphate aldolase, PEP carboxykinase and fumarase, as well as decreased activity of pyruvate kinase, pyruvate formate-lyase, and acetate kinase explained the enhanced succinic acid production and decreased acetic acid formation. Metabolic flux analysis suggested that increased flux to NADH was the main reason for increased activity of the C4 pathway resulting in increased yields of succinic acid. The present work will be propitious to the development of a bio-succinic acid fermentation industry.

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.

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.

Classification of avian haemolytic Actinobacillus-like organisms (Bisgaard taxon 26) associated with anseriforme birds as Actinobacillus anseriformium sp. nov.

Avian haemolytic Actinobacillus-like organisms have tentatively been named Bisgaard taxon 26. Phenotypic information has been published on 65 strains of this taxon. In the current study, 31 isolates were selected for genotypic characterization. Thirty strains had the same rpoB sequence and only one strain diverged in 1 nt. The highest rpoB similarity to members of other taxa was 89.7 % to the type strain of Actinobacillus equuli subsp. haemolyticus and the similarity to the type strain of the type species, Actinobacillus lignieresii, was 88.2 %. The lowest 16S rRNA gene sequence similarity between strains of the group was determined in previous investigations to be 99.6 % and the highest similarities of 96.4 and 96.2 % outside the group were obtained to the reference strain of Actinobacillus genomospecies 2 and to the type strain of A. equuli subsp. equuli, respectively; 95.8-95.3 % similarity was obtained with the type strain of A. lignieresii. recN gene sequence similarities within the group were from 99.5 % (strains F66(T) and F64) to 99.8 % (strains F66(T) and F67) corresponding to genome similarities of 93.9-94.6 %, which are near the upper limit for species compared with other members of the Pasteurellaceae. The highest recN similarity outside the group (83.4 %) was observed to the type strain of Actinobacillus capsulatus, whereas the similarity to the type strain of A. lignieresii was 80.9 %, corresponding to genome similarities of 57.7 and 52.0 %, respectively. All isolates meet the phenotypic characters outlined for Actinobacillus (urease-, phosphatase- and porphyrin-positive, indole-negative, acid production from fructose, sucrose, maltose and dextrin). ß-Haemolysis of bovine blood is observed and isolates may demonstrate in vitro satellitic growth, referred to as V-factor or NAD requirement. Isolates have been obtained from the upper respiratory tract of web-footed birds in which they may cause sinusitis, conjunctivitis and septicaemia. Based on the characterization reported, it is proposed that the isolates belong to a novel species, Actinobacillus anseriformium sp. nov., which includes taxon 26 and a V-factor-dependent strain. The major fatty acids of the type strain are C(16 : 1)ω7c, C(14 : 0), C(16 : 0) and C(14 : 0) 3-OH and/or iso-C(16 : 1) I, corresponding to the profile observed for the type strain of A. lignieresii. Five to 12 characters separate A. anseriformium from other taxa of Actinobacillus, with Actinobacillus ureae being most closely related; A. anseriformium can be differentiated from A. ureae based on haemolysis, ß-glucosidase, and production of acid from (-)-D-sorbitol, trehalose and glycosides. The type strain of A. anseriformium is F66(T) ( = CCUG 60324(T) = CCM 7846(T)), which was isolated from conjunctivitis in a White Pekin duck.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome.

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by Actinobacillus succinogenes.

Succinic acid, a four-carbon diacid, has been the focus of many research projects aimed at developing more economically viable methods of fermenting sugar-containing natural materials. Succinic acid fermentation processes also consume CO(2), thereby potentially contributing to reductions in CO(2) emissions. Succinic acid could also become a commodity used as an intermediate in the chemical synthesis and manufacture of synthetic resins and biodegradable polymers. Much attention has been given recently to the use of microorganisms to produce succinic acid as an alternative to chemical synthesis. We have attempted to maximize succinic acid production by Actinobacillus succinogenes using an experimental design methodology for optimizing the concentrations of the medium components. The first experiment consisted of a 2(4-1) fractional factorial design, and the second entailed a Central Composite Rotational Design so as to achieve optimal conditions. The optimal concentrations of nutrients predicted by the model were: NaHCO(3), 10.0 g l(-1); MgSO(4), 3.0 g l(-1); yeast extract, 2.0 g l(-1); KH(2)PO(4). 5.0 g l(-1); these were experimentally validated. Under the best conversion conditions, as determined by statistical analysis, the production of succinic acid was carried out in an instrumented bioreactor using sugarcane bagasse hemicellulose hydrolysate, yielding a concentration of 22.5 g l(-1).

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.

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.