A repertoire of alginate lyases in the alginate polysaccharide utilization loci of marine bacterium Wenyingzhuangia fucanilytica: biochemical properties and action pattern.

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T , and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35-50 °C and pH 8.0-9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M-specific enzyme (Aly17A). Ultra-performance liquid chromatography-mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M-blocks of oligosaccharide products, as well as the M-blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry.

A Sexually Dimorphic Olfactory Neuron Mediates Fixed Action Transition during Courtship Ritual in Drosophila melanogaster.

Animals perform a series of actions in a fixed order during ritualistic innate behaviors. Although command neurons and sensory pathways responding to external stimuli that trigger these behaviors have been identified, how each action is induced in a fixed order in response to multimodal sensory stimuli remains unclear. Here, the sexually dimorphic lateral antennal lobe tract projection neuron 4 (lPN4) in male Drosophila melanogaster mediates the expression of a fixed behavioral action pattern at the beginning of the courtship ritual, in which a male taps a female body and then extends a wing unilaterally to produce a courtship song. We found that blocking the synaptic output of lPN4 caused an increase in the ratio of male flies that extended a wing unilaterally without tapping the female body, whereas excitation of lPN4 suppressed the transition from the tapping phase to the unilateral wing extension phase. Real-time calcium imaging showed that lPN4 is activated by a volatile pheromone, palmitoleic acid, whose responses were inhibited by simultaneous gustatory stimulation with female cuticular hydrocarbons, showing the existence of an "AND-gate" for multimodal sensory inputs during male courtship behaviors. These results suggest that the function of lPN4 is to suppress unilateral wing extension while responding to a female smell, which is released by appropriate contact chemosensory inputs received when tapping a female. As the female smell also promotes male courtship behaviors, the olfactory system is ready to simultaneously promote and suppress the progress of courtship actions while responding to a female smell.SIGNIFICANCE STATEMENT Although it has been 80 years since Konrad Lorenz and Niko Tinbergen introduced how multiple acts comprising separate innate behaviors are released in a fixed order according to external stimuli, the neural circuits responsible for such fixed action patterns remain largely unknown. The male courtship behavior of Drosophila melanogaster is a good model to investigate how such a fixed behavioral sequence is determined in the brain. Here, we show that lateral antennal lobe tract projection neuron 4 (lPN4) in D. melanogaster functions as an "AND-gate" for volatile and contact chemosensory inputs, mediating the expression of tapping behaviors before unilateral wing extension during male courtship rituals.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823-7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAß1-3GalNAc(4S) or GlcUAß1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAß1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)ß1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Bacterial 1,4-α-glucan branching enzymes: characteristics, preparation and commercial applications.

The 1,4-α-glucan branching enzymes (GBEs) are ubiquitously distributed in animals, microorganisms and plants. These enzymes modify the structure of both starch and glycogen; changing the frequency and position of branches by forming new α-1,6-glucosidic linkages. In organisms, controlling the number and distribution of branches is an irreplaceable process that maintains the physiological state of starch and glycogen in the cell. The process is also the foundation for the industrial applications of GBEs. So far, a number of GBEs have been identified in eukaryotes and prokaryotes as researchers searched for GBEs with optimal properties. Among them, bacterial GBEs have received particular attention due to the convenience of heterologous expression and industrial applications of GBEs from bacteria than GBEs from other sources. The advantages of bacterial GBEs in potential applications stimulated the investigations of bacterial GBEs in terms of their structure and properties. However, full exploitation of GBEs in commercial applications is still in its infancy because of the disadvantages of currently available enzymes and of limited imagination with respect to future possibilities. Thus, in this review, we present an overview of the bacterial GBEs including their structure, biochemical properties and commercial applications in order to depict the whole picture of bacterial GBEs.

Specificity and action pattern of heparanase Bp, a ß-glucuronidase from Burkholderia pseudomallei.

The specificity and action pattern of a ß-glucuronidase derived from the pathogenic bacteria Burkholderia pseudomallei and expressed in Escherichia coli as a recombinant protein has been evaluated. While this enzyme shows activity on a number of glycosaminoglycans, our study has focused on its action on heparin, heparan sulfate and their biosynthetic intermediates as well as chemoenzymatically synthesized, structurally defined heparan sulfate oligosaccharides. These heparin/heparan sulfate (HP/HS) substrates examined varied in size and structure, but all contained an uronic acid (UA) residue ß-(1→4) linked to a glucosamine residue. On the substrates tested, this enzyme (heparanase Bp) acted only on a glucuronic acid residue ß-(1→4) linked to an N-acetylglucosamine, N-sulfoglucosamine or N-acetyl-6-O-sulfoglucosamine residue. A substrate was required to have a length of pentasaccharide or longer and heparanase Bp acted with a random endolytic action pattern on HP/HS. The specificity and glycohydrolase mechanism of action of heparanase Bp resembles mammalian heparanase and is complementary to the bacterial heparin lyases, which act through an eliminase mechanism on a glucosamine residue (1→4) linked to a UA residue, suggesting its utility as a tool for the structural determination of HP/HS as well as representing a possible model for the medically relevant mammalian heparanase. The utility heparanase Bp was demonstrated by the oligosaccharide mapping of heparin, which afforded resistant intact highly sulfated domains ranging from tetrasaccharide to >28-mer with a molecular weight >9000.

Expanding our horizons: central pattern generation in the context of complex activity sequences.

Central pattern generators (CPGs) are central nervous system (CNS) networks that can generate coordinated output in the absence of patterned sensory input. For decades, this concept was applied almost exclusively to simple, innate, rhythmic movements with essentially identical cycles that repeat continually (e.g. respiration) or episodically (e.g. locomotion). But many natural movement sequences are not simple rhythms, as they include different elements in a complex order, and some involve learning. The concepts and experimental approaches of CPG research have also been applied to the neural control of complex movement sequences, such as birdsong, though this is not widely appreciated. Experimental approaches to the investigation of CPG networks, both for simple rhythms and for complex activity sequences, have shown that: (1) brief activation of the CPG elicits a long-lasting naturalistic activity sequence; (2) electrical stimulation of CPG elements alters the timing of subsequent cycles or sequence elements; and (3) warming or cooling CPG elements respectively speeds up or slows down the rhythm or sequence rate. The CPG concept has also been applied to the activity rhythms of populations of mammalian cortical neurons. CPG concepts and methods might further be applied to a variety of fixed action patterns typically used in courtship, rivalry, nest building and prey capture. These complex movements could be generated by CPGs within CPGs ('nested' CPGs). Stereotypical, non-motor, non-rhythmic neuronal activity sequences may also be generated by CPGs. My goal here is to highlight previous applications of the CPG concept to complex but stereotypical activity sequences and to suggest additional possible applications, which might provoke new hypotheses and experiments.

Unexpected mode of action of sweet potato ß-amylase on maltooligomer substrates.

ß-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato ß-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme-product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.

Action Pattern of a Novel G-Specific Alginate Lyase: Determination of Subsite Specificity by HPAEC-PAD/MS.

G-specific alginate lyases are important tools for alginate fragment biodegradation and oligosaccharide production, which have great potential in alginate refining research. In this research, a novel G-specific alginate lyase Aly7Ce was cloned, expressed, and characterized, with the optimal reaction conditions at 30 °C and pH 8.0. By employing the UPSEC-VWD-MS method, Aly7Ce was confirmed as a random endoacting alginate lyase. Its minimum substrate was tetrasaccharide, and the final product majorly consisted of disaccharide to tetrasaccharide. HPAEC-PAD/MS method was employed to investigate the structurally different unsaturated alginate oligosaccharides. The substrate recognition and subsite specificity of Aly7Ce were revealed by detecting the oligosaccharide pattern in the enzymatic products with oligosaccharides or polysaccharides as substrates. Aly7Ce mainly attacked the second glycosidic linkage from the nonreducing end of oligosaccharide substrates. The subsite specificity of Aly7Ce was revealed as -2 (M/G), - 1 (G), + 1 (M/G), and +2 (M/G). The regular oligosaccharide products of Aly7Ce could be applied for the efficient preparation of ΔG, ΔGG, and ΔGGG with high purity. The G-specific alginate lyase Aly7Ce with a well-defined product composition and action pattern provided a novel tool for the modification and structural elucidation of alginate, as well as for the targeted preparation of oligosaccharides.

Discovery and characterization of a novel poly-mannuronate preferred alginate lyase: The first member of a new polysaccharide lyase family.

Alginate is one of the most important marine colloidal polysaccharides, and its oligosaccharides have been proven to possess diverse biological functions. Alginate lyases could specifically degrade alginate and therefore serve as desirable tools for the research and development of alginate. In this report, a novel catalytic domain, which demonstrated no significant sequence similarity with all previously defined functional domains, was verified to exhibit a random endo-acting lyase activity to alginate. The action pattern analysis revealed that the heterologously expressed protein, named Aly44A, preferred to degrade polyM. Its minimum substrates and the minimum products were identified as unsaturated alginate trisaccharides and disaccharides, respectively. Based on the sequence novelty of Aly44A and its homologs, a new polysaccharide lyase family (PL44) was proposed. The discovery of the novel enzyme and polysaccharide lyase family provided a new entrance for the gene-mining and acquiring of alginate lyases, and would facilitate to the utilization of alginate and its oligosaccharides.

Peptide-driven control of somersaulting in Hydra vulgaris.

The cnidarian Hydra vulgaris has a simple nervous system with a few hundred neurons in distributed networks. Yet Hydra can perform somersaults, a complex acrobatic locomotion. To understand the neural mechanisms of somersaulting we used calcium imaging and found that rhythmical potential 1 (RP1) neurons activate before somersaulting. Decreasing RP1 activity or ablating RP1 neurons reduced somersaulting, while two-photon activation of RP1 neurons induced somersaulting. Hym-248, a peptide synthesized by RP1 cells, selectively generated somersaulting. We conclude that RP1 activity, via release of Hym-248, is necessary and sufficient for somersaulting. We propose a circuit model to explain the sequential unfolding of this locomotion, using integrate-to-threshold decision making and cross-inhibition. Our work demonstrates that peptide-based signaling is used by simple nervous systems to generate behavioral fixed action patterns. VIDEO ABSTRACT.

Analysis of the action pattern of sequential α-amylases from B. stearothermophilus and B. amyloliquefaciens on highly concentrated soluble starch.

Hydrolysis of highly concentrated soluble starch (60%, w/w) was performed using sequential α-amylases from Bacillus stearothermophilus (T, 0.2%, w/w) and Bacillus amyloliquefaciens (B, 0.1%, w/w) to identify their possible action patterns. We found that T reduced the average molecular weight (Mw) of soluble starch from 52,827 Da to 31,914 Da and significantly affected its branched chain length. Compared with soluble starch, the chains with DP 6-12 and DP ≥ 13 in the T samples were diminished by 46% and 96%, respectively. This resulted in an attenuation in the proportions of exterior and inner chains, as well as low iodine binding capacity of the hydrolysates. In contrast, a slower decrease in the average Mw of soluble starch occurred after TB incubation, and the level of DP 6-12 further lowered, causing a gradual decline in the iodine binding capacity of the hydrolysates. Gathered data revealed an unusual action pattern of sequential α-amylase treatment at high substrate concentrations. Bacillus stearothermophilus α-amylase exhibited more pronounced endo-hydrolysis of amylopectin, whereas the attack of Bacillus amyloliquefaciens α-amylase on the exterior chains was enhanced in amylopectin residues. These findings suggest that the synergy of various α-amylases is an effective strategy to promote the dextrinization of highly concentrated starch and finely modify the molecular structure of starch.

Documenting Aggression, Dominance and the Impacts of Visitor Interaction on Galápagos Tortoises (Chelonoidis nigra) in a Zoo Setting.

Ensuring high levels of welfare is imperative for modern zoos, but such organisations must also engage visitors in order to successfully spread awareness and raise conservation funds. It is therefore important to understand the responses of animals to visitor interaction to optimise welfare. Often, the opportunity to interact with humans may be enriching for animals, but in other contexts, this interaction may have negative welfare effects. We observed captive female Galápagos giant tortoises (Chelonoidis nigra) to describe aggressive interactions, characterize hierarchy using Elo ratings and assess the impact of visitor interactions. Elo ratings indicated that one individual was dominant over two equally ranked subordinates; aggressive interactions are discussed in this context. We detected significant effects of the presence of visitors and visitor type (keepers, vets or public) within the enclosure on aggression and activity. We suggest that previous miscategorisation of a natural behaviour (the finch response) as an operantly conditioned behaviour, rather than a fixed action pattern, may have triggered aggression. We then document changes made to the management of the animals to mitigate the impacts discovered. This work highlights the importance of empirical evidence in determining optimal management strategies for zoo animals with regards to public interactions and animal welfare.

Neuroethology of acoustic communication in field crickets - from signal generation to song recognition in an insect brain.

Field crickets are best known for the loud calling songs produced by males to attract conspecific females. This review aims to summarize the current knowledge of the neurobiological basis underlying the acoustic communication for mate finding in field crickets with emphasis on the recent research progress to understand the neuronal networks for motor pattern generation and auditory pattern recognition of the calling song in Gryllus bimaculatus. Strong scientific interest into the neural mechanisms underlying intraspecific communication has driven persistently advancing research efforts to study the male singing behaviour and female phonotaxis for mate finding in these insects. The growing neurobiological understanding also inspired many studies testing verifiable hypotheses in sensory ecology, bioacoustics and on the genetics and evolution of behaviour. Over last decades, acoustic communication in field crickets served as a very successful neuroethological model system. It has contributed significantly to the scientific process of establishing, reconsidering and refining fundamental concepts in behavioural neurosciences such as command neurons, central motor pattern generation, corollary discharge processing and pattern recognition by sensory feature detection, which are basic building blocks of our modern understanding on how nervous systems control and generate behaviour in all animals.

Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides.

BACKGROUND: The alginate oligosaccharides have been widely used in agriculture, medicine, and food industries due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities. The bifunctional alginate lyases can degrade the alginate polysaccharide more efficiently into alginate oligosaccharides. Therefore, it is crucial to discover new bifunctional alginate lyase for alginate oligosaccharide production. RESULTS: Herein, a novel bifunctional alginate lyase FsAlgB was cloned and identified from deep-sea bacterium Flammeovirga sp. NJ-04, which exhibited broad substrate specificity and the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. Furthermore, the K m values of FsAlgB towards polyG (0.69 mM) and polyMG (0.92 mM) were lower than that towards sodium alginate (1.28 mM) and polyM (2.06 mM). Recombinant FsAlgB was further characterized as an endolytic alginate lyase, and it can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of - 3 and + 1. CONCLUSION: This study provided extended insights into the substrate recognition and degrading pattern of alginate lyases with broad substrate specificity.

Tongue protrusions modify the syntax of skilled reaching for food by the mouse: Evidence for flexibility in action selection and shared hand/mouth central modulation of action.

Skilled reaching for food by the laboratory mouse has the appearance of an action pattern with a distinctive syntax in which ten submovements occur in an orderly sequence. A mouse locates the food by Sniffing, Lifts, Aims, Advances, and Shapes the hand to Pronate it over a food target that it Grasps, Retracts, and Withdraws to Release to its mouth for eating. The structure of the individual actions in the chain are useful for the study of the mouse motor system and contribute to the use of the mouse as a model of human neurological conditions. The present study describes tongue protrusions that modify the syntax of reaching by occurring at the point of the reaching action at which the hand is at the Aim position. Tongue protrusions were not related to reaching success and were not influenced by training. Tongue protrusions were more likely to occur in the presence of a food target than with reaches made when food was absent. There were vast individual differences; some mice always make tongue protrusions while other mice never make tongue protrusions. That the syntax of reaching can be altered by the insertion of a surrogate (co-occurring) movement adds to a growing body of evidence that skilled reaching is assembled from a number of relatively independent actions, each with its own sensorimotor control that are subject to central modulation. That tongue and hand reaching movements can co-occur suggests a privileged relation between neural mechanisms that control movements of the tongue and hand.

Maltooligosaccharide-forming amylase: Characteristics, preparation, and application.

As member of glycosyl hydrolase family 13, maltooligosaccharide-forming amylases (MFAses) are specific and interesting because of their capacity to hydrolyze starch into functional maltooligosaccharides, which are usually composed of 2-10 α-d-glucopyranosyl units linked by α-1,4 glycosidic linkages. MFAses have been extensively studied during recent decades, and have shown promise in various industrial applications. This review begins by introducing the potential uses of maltooligosaccharides. Then it describes the progress in the identification, assay, action pattern, structure, and modification of MFAses. The review continues with tips concerning the preparation of MFAses, which aim to improve MFAse production to meet the needs of industry. Finally, the industrial uses of MFAses are described, focusing on the production of maltooligosaccharides and application in the bread industry. Recent progress has demonstrated that the MFAses are poised to become important industrial catalysts.

Revisiting the concept of behavior patterns in animal behavior with an example from food-caching sequences in wolves (Canis lupus), coyotes (Canis latrans), and red foxes (Vulpes vulpes).

We discuss the history, conceptualization, and relevance of behavior patterns in modern ethology by explaining the evolution of the concepts of fixed action patterns and modal action patterns. We present the movement toward a more flexible concept of natural action sequences with significant degrees of (production and expressive) freedom. An example is presented with the food caching behavior of three Canidae species: red fox (Vulpes vulpes), coyote (Canis latrans) and gray wolf (Canis lupus). Evolutionary, ecological, and neuroecological/neuroethological arguments are presented to explain the difference in levels of complexity and stereotypy between Canis and Vulpes. This article is part of a Special Issue entitled: Canine Behavior.

The cognitive roles of behavioral variability: idiosyncratic acts as the foundation of identity and as transitional, preparatory, and confirmatory phases.

Behavior in obsessive compulsive disorder (OCD), in habitual daily tasks, and in sport and cultural rituals is deconstructed into elemental acts and categorized into common acts, performed by all individuals completing a similar task, and idiosyncratic acts, not performed by all individuals. Never skipped, common acts establish the pragmatic part of motor tasks. Repetitive performance of a few common acts renders rituals a rigid form, whereby common acts may serve as memes for cultural transmission. While idiosyncratic acts are not pragmatically necessary for task completion, they fulfill important cognitive roles. They form a long preparatory phase in tasks that involve high stakes, and a long confirmatory phase in OCD rituals. Idiosyncratic acts also form transitional phases between motor tasks, and are involved in establishing identity and preserving the flexibility necessary for adapting to varying circumstances. Behavioral variability, as manifested in idiosyncrasy, thus does not seem to be a noise or by-product of motor activity, but an essential cognitive component that has been preserved in the evolution of behavioral patterns, similar to the genetic variability in biology.