Polysaccharide Lyase Research Articles

In-silico analysis of the regulatory region of effector protein genes in Verticillium dahliae

Verticillium dahliae is a soil-borne fungal plant pathogen that infects more than 400 plant species throughout the world, causing huge economic losses annually. Due to its wide host range and adaptation strategy of producing resting structures, Verticillium infection and spread are difficult to control. The resting dormant melanized structures called microsclerotia, produced during the dormant stage of the pathogen can persist in soil for years and are the primary source of infection as they germinate after being stimulated by root exudates. The fungus secrets effector proteins that facilitate the primary infection and subsequent spread. In the current study, we used in silico tools to analyze effector proteins belonging to hydrophobin, necrosis, and ethylene inducing like-protein and polysaccharide lyase 3 protein family which are required for virulence and pathogenicity of the pathogen. Our study showed that the genes for these protein families consist of many paralogs. Some of these paralogous proteins display similar functions but differential expression during pathogenicity establishment. This study will provide information about cis-acting factors regulating differential regulation of paralogs during infection.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use

BackgroundK1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge.MethodsWe analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes.ResultsWe found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose.ConclusionsOur results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyases.

Green macroalgae, e.g., Ulva lactuca, are valuable bioactive sources of nutrients; but algae recalcitrant cell walls, composed of a complex cross-linked matrix of polysaccharides, can compromise their utilization as feedstuffs for monogastric animals. This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases to degrade U. lactuca cell walls and release nutritive compounds. A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U. lactuca cell wall polysaccharides. The enzymes were incubated with the macroalga, either alone or in combination, to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls. The individual action of a polysaccharide lyase family 25 (PL25), an ulvan lyase, was shown to be the most efficient in cell wall disruption. The ulvan lyase treatment, in triplicate measures, promoted the release of 4.54 g/L (P < 0.001) reducing sugars, a mono- and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga (P < 0.01), respectively, and a decrease of 41.7% (P < 0.001) in cell wall fluorescence, in comparison to control. The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated. A release of some monounsaturated fatty acids was observed, particularly the health beneficial 18:1c9 (P < 0.001). However, no significant release of total fatty acids (P > 0.05), proteins (P = 0.861) or pigments (P > 0.05) was found. These results highlight the capacity of a single recombinant ulvan lyase (PL25 family) to incompletely disrupt U. lactuca cell walls. This enzyme could enhance the bioaccessibility of U. lactuca bioactive products with promising utilization in the feed industry.

The Function of CBM32 in Alginate Lyase VxAly7B on the Activity on Both Soluble Sodium Alginate and Alginate Gel.

Carbohydrate-binding modules (CBMs), as an important auxiliary module, play a key role in degrading soluble alginate by alginate lyase, but the function on alginate gel has not been elucidated. Recently, we reported alginate lyase VxAly7B containing a CBM32 and a polysaccharide lyase family 7 (PL7). To investigate the specific function of CBM32, we characterized the full-length alginate lyase VxAly7B (VxAly7B-FL) and truncated mutants VxAly7B-CM (PL7) and VxAly7B-CBM (CBM32). Both VxAly7B-FL and native VxAly7B can spontaneously cleavage between CBM32 and PL7. The substrate-binding capacity and activity of VxAly7B-CM to soluble alginate were 0.86- and 1.97-fold those of VxAly7B-FL, respectively. Moreover, CBM32 could accelerate the expansion and cleavage of alginate gel beads, and the degradation rate of VxAly7B-FL to alginate gel beads was threefold that of VxAly7B-CM. Results showed that CBM32 is not conducive to the degradation of soluble alginate by VxAly7B but is helpful for binding and degradation of insoluble alginate gel. This study provides new insights into the function of CBM32 on alginate gel, which may inspire the application strategy of CBMs in insoluble substrates.

Identification and Expression of Some Plant Cell Wall-Degrading Enzymes Present in Three Ontogenetics Stages of Thecaphora frezii, a Peanut (&amp;lt;i&amp;gt;Arachis hypogaea&amp;lt;/i&amp;gt; L.) Pathogenic Fungus

Peanuts can be affected by the presence of pathogenic microorganisms. The fungus Thecaphora frezii (T. frezii), which belongs to the taxonomic class Ustilaginomycetes, is the causal agent of the disease known as “peanut smut”. The life cycle of this fungus includes three stages, namely teliospores, basidiospores and hyphae. In the hyphae stage, infection occurs in the peanut plant, which requires the involvement of some enzymes secreted by the fungus. These include the Plant Cell Wall-Degrading Enzymes (PCWDEs), which degrade various polysaccharides. This study aimed to identify the presence of transcript for enzymes belonging to the PCWDEs from three stages of T. frezii. For this, total RNA was extracted from the three ontogenetic stages of T. frezii. These samples were analyzed using an RNA-Seq approach and some transcripts were quantified using Real Time PCR. The analysis of the data provided by the RNA-Seq of the three T. frezii stages, it was possible to identify some transcripts that could encode enzymes compatible with polysaccharides degradation that are part of the plant cell wall. In T. frezii transcriptome, 40 deduced proteins would be enzymes with functions of PCWDEs were identified. They were divided into 27 glycoside hydrolases; two polysaccharide lyases; three carbohydrate esterases and eight enzymes with auxiliary activities. In addition, the fungal SNF1 gene was identified whose activity could be affected by high glucose level, and indirectly influence the levels of some PCWDEs. The analysis of the PCWDEs could help to understand part of the fungal infection process and possibly find substances that can control its development.

Whole genome sequencing of an edible and medicinal mushroom, Russula griseocarnosa, and its association with mycorrhizal characteristics.

Russula griseocarnosa is a well-known ectomycorrhizal mushroom, which is mainly distributed in the Southern China. Although several scholars have attempted to isolate and cultivate fungal strains, no accurate method for culture of artificial fruiting bodies has been presented owing to difficulties associated with mycelium growth on artificial media. Herein, we sequenced R. griseocarnosa genome using the second- and third-generation sequencing technologies, followed by de novo assembly of high-throughput sequencing reads, and GeneMark-ES, BLAST, CAZy, and other databases were utilized for functional gene annotation. We also constructed a phylogenetic tree using different species of fungi, and also conducted comparative genomics analysis of R. griseocarnosa against its four representative species. In addition, we evaluated the accuracy of one already sequenced genome of R. griseocarnosa based on the internal transcribed spacer (ITS) sequencing of that type of species. The assembly process resulted in identification of 230 scaffolds with a total genome size of 50.67 Mbp. The gene prediction showed that R. griseocarnosa genome included 14,229 coding sequences (CDs). In addition, 470 RNAs were predicted with 155 transfer RNAs (tRNAs), 49 ribosomal RNAs (rRNAs), 41 small noncoding RNAs (sRNAs), 42 small nuclear RNAs (snRNAs), and 183 microRNAs (miRNAs). The predicted protein sequences of R. griseocarnosa were analyzed to indicate the existence of carbohydrate-active enzymes (CAZymes), and the results revealed that 153 genes encoded CAZymes, which were distributed in 58 CAZyme families. These enzymes included 78 glycoside hydrolases (GHs), 34 glycosyl transferases (GTs), 30 auxiliary activities (AAs), 2 carbohydrate esterases (CEs), 8 carbohydrate-binding modules (CBMs), and only one polysaccharide lyase (PL). Compared with other fungi, R. griseocarnosa had fewer CAZymes, and the number and distribution of CAZymes were similar to other mycorrhizal fungi, such as Tricholoma matsutake and Suillus luteus. Well-defined effector proteins that were associated with mycorrhiza-induced small-secreted proteins (MiSSPs) were not found in R. griseocarnosa, which indicated that there may be some special effector proteins to interact with host plants in R. griseocarnosa. The genome of R. griseocarnosa may provide new insights into the energy metabolism of ectomycorrhizal (ECM) fungi, a reference to study ecosystem and evolutionary diversification of R. griseocarnosa, as well as promoting the study of artificial domestication.

Cloning, expression, and characterization of a glycosaminoglycan lyase from Vibrio sp. H240

Glycosaminoglycan lyase is an effective tool for the functional studies of glycosaminoglycans and for the preparation of oligosaccharides. In this study, a new glycosaminoglycan lyase HCLaseV with a molecular weight of 90 kDa was cloned, expressed, and characterized from Vibrio sp. H240. The lyase belonged to the polysaccharide lyase (PL)− 8 family. HCLaseV showed enzyme activities toward chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, and hyaluronic acid. HCLaseV exhibited the highest activity against HA at pH 7.0 and 40 °C. HCLaseV was an endo-type enzyme whose degradation end-product was unsaturated disaccharides. Ca2+ inhibited the activity of HCLaseV to a certain extent, which was different from most of the enzymes in the PL-8 family. Mutagenesis studies showed that the Ca2+ inhibition might be related to the Asn244 residue. The sequence homology was evaluated by mutagenesis studies, and the catalytic residues in HCLaseV were presumed to be His278, Trp485, and Tyr287. These characteristics are helpful for further basic research and application.

Responses of microbial function, biomass and heterotrophic respiration, and organic carbon in fir plantation soil to successive nitrogen and phosphorus fertilization.

Carbon dioxide (CO2) emissions from forest ecosystems originate largely from soil respiration, and microbial heterotrophic respiration plays a critical role in determining organic carbon (C) stock. This study investigated the impacts of successive nitrogen (N) and phosphorus (P) fertilization after 9years on soil organic C stock; CO2 emission; and microbial biomass, community, and function in a Chinese fir plantation. The annual fertilization rates were (1) CK, control without N or P fertilization; (2) N50, 50kgNha-1; (3) N100, 100kgNha-1; (4) P50, 50kgP ha-1; (5) N50P50, 50kgNha-1 + 50kgPha-1; and (6) N100P50, 100kgNha-1 + 50kgPha-1. The N100P50 treatment had the highest cumulative soil CO2 emissions, but the CK treatment had the lowest cumulative soil CO2 emissions among all treatments. The declines of soil organic C (SOC) after successive 9-year fertilization were in the order of 100kgNha-1year-1 > 50kgNha-1year-1 > CK. Compared to the CK treatment, successive N fertilization significantly changed soil microbial communities at different application rates and increased the relative gene abundances of glycoside hydrolases, glycosyl transferases, carbohydrate-binding modules, and polysaccharide lyases at 100kgNha-1year-1. Relative to P fertilization alone (50kgPha-1year-1), combined N and P fertilization significantly altered the soil microbial community structure and favored more active soil microbial metabolism. Microbial community and metabolism changes caused by N fertilization could have enhanced CO2 emission from heterotrophic respiration and eventually led to the decrease in organic C stock in the forest plantation soil. KEY POINTS: • N fertilization, alone or with P, favored more active microbial metabolism genes. • 100kgNha-1 fertilization significantly changed microbial community and function. • N fertilization led to a "domino effect" on the decrease of soil C stock.

Discovery of a Novel Glucuronan Lyase System in Trichoderma parareesei.

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear β-(1,4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei. The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. Nuclear magnetic resonance revealed that the monomeric end product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and high-performance anion-exchange chromatography with pulsed amperometric detection analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales species with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Trichoderma parareseei. Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareesei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed.

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.

Evolving strategies for marine enzyme engineering: recent advances on the molecular modification of alginate lyase.

Alginate, an acidic polysaccharide, is formed by β-d-mannuronate (M) and α-l-guluronate (G). As a type of polysaccharide lyase, alginate lyase can efficiently degrade alginate into alginate oligosaccharides, having potential applications in the food, medicine, and agriculture fields. However, the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature stability. In recent years, various structural features of alginate lyase have been determined, resulting in modification strategies that can increase the applicability of alginate lyase, making it important to summarize and discuss the current evidence. In this review, we summarized the structural features and catalytic mechanisms of alginate lyase. Molecular modification strategies, such as rational design, directed evolution, conserved domain recombination, and non-catalytic domain truncation, are also described in detail. Lastly, the application of alginate lyase is discussed. This comprehensive summary can inform future applications of alginate lyases.

Whole-Genome Data from Curtobacterium flaccumfaciens pv. flaccumfaciens Strains Associated with Tan Spot of Mungbean and Soybean Reveal Diverse Plasmid Profiles.

Despite the substantial economic impact of Curtobacterium flaccumfaciens pv. flaccumfaciens on legume production worldwide, the genetic basis of its pathogenicity and potential host association is poorly understood. The production of high-quality reference genome assemblies of C. flaccumfaciens pv. flaccumfaciens strains associated with different hosts sheds light on the genetic basis of its pathogenic variability and host association. Moreover, the study of recent outbreaks of bacterial wilt and microevolution of the pathogen in Australia requires access to high-quality reference genomes that are sufficiently closely related to the population being studied within Australia. We provide the first genome assemblies of C. flaccumfaciens pv. flaccumfaciens strains associated with mungbean and soybean, which revealed high variability in their plasmid composition. The analysis of C. flaccumfaciens pv. flaccumfaciens genomes revealed an extensive suite of carbohydrate-active enzymes potentially associated with pathogenicity, including four carbohydrate esterases, 50 glycoside hydrolases, 23 glycosyl transferases, and a polysaccharide lyase. We also identified 11 serine peptidases, three of which were located within a linear plasmid, pCff119. These high-quality assemblies and annotations will provide a foundation for population genomics studies of C. flaccumfaciens pv. flaccumfaciens in Australia and for answering fundamental questions regarding pathogenicity factors and adaptation of C. flaccumfaciens pv. flaccumfaciens to various hosts worldwide and, at a broader scale, contribute to unraveling genomic features of gram-positive, xylem-inhabiting bacterial pathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Антибиопленочная активность антиматриксных молекул

Внеклеточный матрикс биопленок обеспечивает фиксацию биопленки на биологической поверхности и защиту ее бактерий от внешних неблагоприятных факторов. Основным структурным компонентом био­пленок является внеклеточное полисахаридное вещество. Ключевыми молекулярными структурами, поддерживающими трехмерную структуру биопленки, считают экзополисахариды и амилоидоподобные волокна. До недавнего времени предполагалось, что большинство механизмов диспергирования биопленок ассоциировано с функционированием матриксных деградирующих ферментов, таких как гликозидгидролазы, полисахаридные лиазы и протеазы. Однако продемонстрировано, что в процессе разрушения матриксных экзополисахаридов и амилоидоподобных волокон играют самостоятельную роль малые молекулы. Среди соединений, нарушающих матрикс биопленки, различают антиматриксные молекулы, соединения, взаимодействующие с микродоменами бактериальной мембраны, и бактериальные поверхностно-активные вещества (биосурфагенты). Продемонстрировано, что соединения данных групп могут ингибировать образование биопленок и способствовать их диспергированию. Из группы антиматриксных молекул полиаминное соединение норспермидин взаимодействует с экзополисахаридами, а производные бензохинона AA-861 и сесквитерпеновый лактон партенолид — с TasA-амилоидоподобными волокнами. Норспермидин предотвращает образование и диспергирует биопленки различных бактерий, включая Acinetobacter baumannii, Bacillus subtili, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis. Соединение AA-861 проявляет активность против биопленок, которые образованы бактериями Streptococcus mutans, Bacillus cereus, Escherichia coli, а партенолид диспергирует биопленки, сформированные Escherichia coli и Bacillus cereus. Сарагосовые кислоты, взаимодействуя с микродоменами бактериальной мембраны, нарушают функционирование рафт-ассоциированных протеинов бактерий. Малые антиматриксные молекулы и нацеленные на микродомены мембраны бактерий, инициирующие диспергирование биопленки, безусловно, станут основанием для разработки эффективных антибиопленочных терапевтических лекарственных средств.

Exploring polysaccharide lyases of PL-5 family through the lens of structure, function and dynamics

Exploring polysaccharide lyases of PL-5 family through the lens of structure, function and dynamics

Structural insights into the mechanism of pH-selective substrate specificity of the polysaccharide lyase Smlt1473

Polysaccharide lyases (PLs) are a broad class of microbial enzymes that degrade anionic polysaccharides. Equally broad diversity in their polysaccharide substrates has attracted interest in biotechnological applications such as biomass conversion to value-added chemicals and microbial biofilm removal. Unlike other PLs, Smlt1473 present in the clinically relevant Stenotrophomonas maltophilia strain K279a demonstrates a wide range of pH-dependent substrate specificities toward multiple, diverse polysaccharides: hyaluronic acid (pH 5.0), poly-β-D-glucuronic (celluronic) acid (pH 7.0), poly-β-D-mannuronic acid, and poly-α-L-guluronate (pH 9.0). To decode the pH-driven multiple substrate specificities and selectivity in this single enzyme, we present the X-ray structures of Smlt1473 determined at multiple pH values in apo and mannuronate-bound states as well as the tetra-hyaluronate-docked structure. Our results indicate that structural flexibility in the binding site and N-terminal loop coupled with specific substrate stereochemistry facilitates distinct modes of entry for substrates having diverse charge densities and chemical structures. Our structural analyses of wild-type apo structures solved at different pH values (5.0–9.0) and pH-trapped (5.0 and 7.0) catalytically relevant wild-type mannuronate complexes (1) indicate that pH modulates the catalytic microenvironment for guiding structurally and chemically diverse polysaccharide substrates, (2) further establish that molecular-level fluctuation in the enzyme catalytic tunnel is preconfigured, and (3) suggest that pH modulates fluctuations resulting in optimal substrate binding and cleavage. Furthermore, our results provide key insight into how strategies to reengineer both flexible loop and regions distal to the active site could be developed to target new and diverse substrates in a wide range of applications.

Genomic insight for algicidal activity in Rhizobium strain AQ_MP.

Occurrence of Harmful Algal Blooms (HABs) creates a threat to aquatic ecosystem affecting the existing flora and fauna. Hence, the mitigation of HABs through an eco-friendly approach remains a challenge for environmentalists. The present study provides the genomic insights of Rhizobium strain AQ_MP, an environmental isolate that showed the capability of degrading Microcystis aeruginosa (Cyanobacteria) through lytic mechanisms. Genome sequence analysis of Rhizobium strain AQ_MP unraveled the algal lytic features and toxin degradative pathways in it. Functional genes of CAZymes such as glycosyltransferases (GT), glycoside hydrolases (GH), polysaccharide lyases (PL) which supports algal polysaccharide degradation (lysis) were present in Rhizobium strain AQ_MP. Genome analysis also clarified the presence of the glutathione metabolic pathway, which is the biological detoxification pathway responsible for toxin degradation. The conserved region mlrC, a microcystin toxin-degrading gene was also annotated in the genome. The study illustrated that Rhizobium strain AQ_MP harbored a wide range of mechanisms for the lysis of Microcystis aeruginosa cells and its toxin degradation. In future, this study finds promiscuity for employing Rhizobium strain AQ_MP species for bioremediation, based on its physiological and genomic analysis.

Massilia cellulosiltytica sp. nov., a novel cellulose-degrading bacterium isolated from rhizosphere soil of rice (Oryza sativa L.) and its whole genome analysis

A bacterial strain, Gram-stain negative, rod-shaped, aerobic and cellulose-degrading, designated NEAU-DD11T, was isolated from rhizosphere soil of rice collected from Northeast Agricultural University in Harbin, Heilongjiang Province, North-east China. Base on 16S rRNA gene sequence analysis, strain NEAU-DD11T belongs to the genus Massilia and shared high sequence similarities with Massilia phosphatilytica 12-OD1T (98.46%) and Massilia putida 6NM-7T (98.41%). Phylogenetic analysis based on the 16S rRNA gene and whole genome sequences indicated that strain NEAU-DD11T formed lineage related to M. phosphatilytica 12-OD1T and M. putida 6NM-7T. The major fatty acids of the strain were C16:0, C17:0-cyclo and C16:1ω7c. The respiratory quinone was Q-8. The polar lipids profile of the strain showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid and an unidentified phospholipid. In addition, the digital DNA-DNA hybridization values between strain NEAU-DD11T and M. phosphatilytica 12-OD1T and M. putida 6NM-7T were 45.4 and 35.6%, respectively, which are lower than the accepted threshold value of 70%. The DNA G + C content of strain NEAU-DD11T was 66.2%. The whole genome analysis showed the strain contained carbohydrate enzymes such as glycoside hydrolase and polysaccharide lyase, which enabled the strain to have the function of degrading cellulose. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, we conclude that strain NEAU-DD11T represents a novel species of the genus Massilia, for which the name Massilia cellulosiltytica sp. nov. is proposed. The type strain is NEAU-DD11T (= CCTCC AB 2019141T = DSM 109721T).

Actinoplanes aureus sp. nov., a novel protease-producing actinobacterium isolated from soil.

A novel protease-producing actinobacterium, designated strain NEAU-A11T, was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China, and characterised using a polyphasic approach. The hydrolytic enzymes, such as proteases, played critical roles in destruction of fungi by degrading the protein linkages to disrupt integrity in the cell wall. This suggested that the isolate could be a good biocontrol candidate against pathogens to control fungal diseases. On the basis of 16S rRNA gene sequence analysis, strain NEAU-A11T was indicated to belong to the genus Actinoplanes and was most closely related to Actinoplanes rectilineatus JCM 3194T (98.9%). Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were arabinose, xylose and glucose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major fatty acids were C18:0, C16:0, C18:1 ω9c, C17:0 and C15:0. Genome sequencing revealed a genome size of 10,742,096bp, a G + C content of 70.5% and 9,514 protein-coding genes (CDS), including 102 genes coding for protease. Moreover, Genome analysis showed that strain NEAU-A11T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Genome mining analysis using antiSMASH 5.0 led to the identification of 20 putative gene clusters responsible for the production of diverse secondary metabolites. Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with A. rectilineatus JCM 3194T in the genus Actinoplanes. Whole-genome phylogeny showed strain NEAU-A11T formed a stable phyletic line with Actinoplanes lutulentus DSM 45883T (97.6%). However, whole-genome average nucleotide identity value between strain NEAU-A11T and its reference strains A. rectilineatus JCM 3194T and A. lutulentus DSM 45883T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24.8% (22.5-27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95-96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G + C in genomic DNA was 70.5%, within the range of 67-76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11T (= CCTCC AA 2019063T = JCM 33971T) as the type strain.

Microbial Regulation of Deterioration and Preservation of Salted Kelp under Different Temperature and Salinity Conditions.

High salinity is an effective measure to preserve kelp, but salted kelp can still deteriorate after long-term preservation. In order to clarify the key conditions and microbial behavior of salted kelp preservation, 10% (S10), 20% (S20), and 30% (S30) salt concentrations were evaluated at 25 °C (T25) and 4 °C (T4). After 30 days storage, these salted kelps showed different states including rot (T25S10), softening (T25S20), and undamaged (other samples). By detecting polysaccharide lyase activity and performing high-throughput sequencing of the prokaryotic 16S rRNA sequence and metagenome, we found that deteriorated kelps (T25S10 and T25S20) had significantly higher alginate lyase activity and bacterial relative abundance than other undamaged samples. Dyella, Saccharophagus, Halomonas, Aromatoleum, Ulvibacter, Rhodopirellula, and Microbulbifer were annotated with genes encoding endonuclease-type alginate lyases, while Bacillus and Thiobacillus were annotated as the exonuclease type. Additionally, no alginate lyase activity was detected in undamaged kelps, whose dominant microorganisms were halophilic archaea without alginate lyase-encoding genes. These results indicated that room-temperature storage may promote salted kelp deterioration due to the secretion of bacterial alginate lyase, while ultra-high-salinity and low-temperature storage can inhibit bacterial alginate lyase and promote the growth of halophilic archaea without alginate lyase, thus achieving the preservation of salted kelp.