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1.
Mar Genomics ; 61: 100911, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35058038

RESUMO

Halomonas sp. MT13, a moderately psychrotolerant, piezotolerant and exopolysaccharide-producing bacterium, was isolated from deep-sea sediment of the Mariana Trench at the depth of 8300 m. Here, we report the complete genome sequence of strain MT13 and its genomic characteristics related to deep-sea environmental adaptation by comparing with its three closely related Halomonas species. The genome of strain MT13 contains one circular chromosome of 3,643,760 bp without any plasmid. Gene annotation, Cluster of Orthologous Groups (COG) and KEGG analysis showed that strain MT13 possesses a serial of genes involved in deep-sea environmental adaptation, including ectoine biosynthesis, osmolyte transport, and cold-shock response. Compared with type strains of three closely related Halomonas species, strain MT13 has higher proportions of genes assigned to translation, ribosomal structure and biogenesis, and coenzyme, lipid and inorganic ion transport and metabolism, but lacks genes involved in flagellar assembly. The genome of strain MT13 would deepen our knowledge on the adaptation strategies of microorganisms dwelling in deep-sea environment.


Assuntos
Halomonas , Aclimatação/genética , Sequência de Bases , Genômica , Halomonas/genética , Filogenia
3.
Appl Environ Microbiol ; : AEM0221921, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34818098

RESUMO

As the most abundant D-amino acid (DAA) in the ocean, D-alanine (D-Ala) is a key component of peptidoglycan in bacterial cell wall. However, the underlying mechanisms of bacterial metabolization of D-Ala through microbial food web remain largely unknown. In this study, the metabolism of D-Ala by marine bacterium Pseudoalteromonas sp. CF6-2 was investigated. Based on genomic, transcriptional and biochemical analyses combined with gene knockout, D-Ala aminotransferase was found to be indispensable for the catabolism of D-Ala in strain CF6-2. Investigation on other marine bacteria also showed that D-Ala aminotransferase gene is a reliable indicator for their ability to utilize D-Ala. Bioinformatic investigation revealed that D-Ala aminotransferase sequences are prevalent in genomes of marine bacteria and metagenomes, especially in seawater samples, and Gammaproteobacteria represents the predominant group containing D-Ala aminotransferase. Thus, Gammaproteobacteria is likely the dominant group to utilize D-Ala via D-Ala aminotransferase to drive the recycling and mineralization of D-Ala in the ocean. IMPORTANCE As the most abundant D-amino acid in the ocean, D-Ala is a component of marine DON (Dissolved organic nitrogen) pool. However, the underlying mechanism of bacterial metabolization of D-Ala to drive the recycling and mineralization of D-Ala in the ocean is still largely unknown. The results in this study showed that D-Ala aminotransferase is specific and indispensable for D-Ala catabolism in marine bacteria, and that marine bacteria containing D-Ala aminotransferase genes are predominantly Gammaproteobacteria widely distributed in global oceans. This study reveals marine D-Ala utilizing bacteria and the mechanism of their metabolization of D-Ala. The results shed light on the mechanisms of recycling and mineralization of D-Ala driven by bacteria in the ocean, which are helpful in understanding oceanic microbial-mediated nitrogen cycle.

4.
Appl Environ Microbiol ; : AEM0180621, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34788071

RESUMO

Dimethylsulfoniopropionate (DMSP) is one of the most abundant organic sulfur compounds in the oceans, which is mainly degraded by bacteria through two pathways, a cleavage pathway and a demethylation pathway. Its volatile catabolites dimethyl sulfide (DMS) and methanethiol (MT) in these pathways play important roles in the global sulfur cycle and have potential influences on the global climate. Intense DMS/DMSP cycling occurs in the Arctic. However, little is known about the diversity of cultivable DMSP-catabolizing bacteria in the Arctic and how they catabolize DMSP. Here, we screened DMSP-catabolizing bacteria from Arctic samples and found that bacteria of four genera (Psychrobacter, Pseudoalteromonas, Alteromonas and Vibrio) could grow with DMSP as the sole carbon source, among which Psychrobacter and Pseudoalteromonas are predominant. Four representative strains (Psychrobacter sp. K31L, Pseudoalteromonas sp. K222D, Alteromonas sp. K632G and Vibrio sp. G41H) from different genera were selected to probe their DMSP catabolic pathways. All these strains produce DMS and MT simultaneously during their growth on DMSP, indicating that all strains likely possess the two DMSP catabolic pathways. On the basis of genomic and biochemical analyses, the DMSP catabolic pathways in these strains were proposed. Bioinformatic analysis indicated that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. This study provides novel insights into DMSP catabolism in marine bacteria. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in the oceans. The catabolism of DMSP is an important step of the global sulfur cycle. Although Gammaproteobacteria are widespread in the oceans, the contribution of Gammaproteobacteria in global DMSP catabolism is not fully understood. Here, we found that bacteria of four genera belonging to Gammaproteobacteria (Psychrobacter, Pseudoalteromonas, Alteromonas and Vibrio), which were isolated from Arctic samples, were able to grow on DMSP. The DMSP catabolic pathways of representative strains were proposed. Bioinformatic analysis indicates that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. Our results suggest that novel DMSP dethiomethylases/demethylases may exist in Pseudoalteromonas, Alteromonas and Vibrio, and that Gammaproteobacteria may be important participants in marine, especially in polar DMSP cycling.

5.
Nat Microbiol ; 6(11): 1351-1356, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34697458

RESUMO

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

6.
Front Microbiol ; 12: 735793, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630359

RESUMO

Dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) are widespread in marine environment, and are important participants in the global sulfur cycle. Microbiol oxidation of DMS to DMSO represents a major sink of DMS in marine surface waters. The SAR11 clade and the marine Roseobacter clade (MRC) are the most abundant heterotrophic bacteria in the ocean surface seawater. It has been reported that trimethylamine monooxygenase (Tmm, EC 1.14.13.148) from both MRC and SAR11 bacteria likely oxidizes DMS to generate DMSO. However, the structural basis of DMS oxidation has not been explained. Here, we characterized a Tmm homolog from the SAR11 bacterium Pelagibacter sp. HTCC7211 (Tmm7211). Tmm7211 exhibits DMS oxidation activity in vitro. We further solved the crystal structures of Tmm7211 and Tmm7211 soaked with DMS, and proposed the catalytic mechanism of Tmm7211, which comprises a reductive half-reaction and an oxidative half-reaction. FAD and NADPH molecules are essential for the catalysis of Tmm7211. In the reductive half-reaction, FAD is reduced by NADPH. In the oxidative half-reaction, the reduced FAD reacts with O2 to form the C4a-(hydro)peroxyflavin. The binding of DMS may repel the nicotinamide ring of NADP+, and make NADP+ generate a conformational change, shutting off the substrate entrance and exposing the active C4a-(hydro)peroxyflavin to DMS to complete the oxidation of DMS. The proposed catalytic mechanism of Tmm7211 may be widely adopted by MRC and SAR11 bacteria. This study provides important insight into the conversion of DMS into DMSO in marine bacteria, leading to a better understanding of the global sulfur cycle.

7.
Microbiome ; 9(1): 207, 2021 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-34654476

RESUMO

BACKGROUND: Dimethyl sulfide (DMS) is the dominant volatile organic sulfur in global oceans. The predominant source of oceanic DMS is the cleavage of dimethylsulfoniopropionate (DMSP), which can be produced by marine bacteria and phytoplankton. Polar oceans, which represent about one fifth of Earth's surface, contribute significantly to the global oceanic DMS sea-air flux. However, a global overview of DMS and DMSP cycling in polar oceans is still lacking and the key genes and the microbial assemblages involved in DMSP/DMS transformation remain to be fully unveiled. RESULTS: Here, we systematically investigated the biogeographic traits of 16 key microbial enzymes involved in DMS/DMSP cycling in 60 metagenomic samples from polar waters, together with 174 metagenome and 151 metatranscriptomes from non-polar Tara Ocean dataset. Our analyses suggest that intense DMS/DMSP cycling occurs in the polar oceans. DMSP demethylase (DmdA), DMSP lyases (DddD, DddP, and DddK), and trimethylamine monooxygenase (Tmm, which oxidizes DMS to dimethylsulfoxide) were the most prevalent bacterial genes involved in global DMS/DMSP cycling. Alphaproteobacteria (Pelagibacterales) and Gammaproteobacteria appear to play prominent roles in DMS/DMSP cycling in polar oceans. The phenomenon that multiple DMS/DMSP cycling genes co-occurred in the same bacterial genome was also observed in metagenome assembled genomes (MAGs) from polar oceans. The microbial assemblages from the polar oceans were significantly correlated with water depth rather than geographic distance, suggesting the differences of habitats between surface and deep waters rather than dispersal limitation are the key factors shaping microbial assemblages involved in DMS/DMSP cycling in polar oceans. CONCLUSIONS: Overall, this study provides a global overview of the biogeographic traits of known bacterial genes involved in DMS/DMSP cycling from the Arctic and Antarctic oceans, laying a solid foundation for further studies of DMS/DMSP cycling in polar ocean microbiome at the enzymatic, metabolic, and processual levels. Video Abstract.


Assuntos
Água do Mar , Oceanos e Mares , Filogenia , Sulfetos , Compostos de Sulfônio
8.
Appl Environ Microbiol ; 87(21): e0152721, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34406825

RESUMO

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. strain SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellum and found that a single-base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281, encoding the core segment and the C-terminal segment, and PSM_A2282, encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin, A2281::82, was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of a normal length. Analyses of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicate that due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete and is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. IMPORTANCE Polar flagella, which are essential organelles for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment, and a C-terminal segment. The results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. strain SM9913 demonstrate that a single-base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.

9.
Biochem Biophys Res Commun ; 569: 1-9, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216991

RESUMO

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor without curable therapy. Surgical resection remains the first choice of patients with GBM but tumors relapse rapidly even combined with conventional chemoradiotherapy. The mechanism of GBM rapid recurrence is poorly understood, which is largely due to the lack of an appropriate animal model, thus heavily impedes the improvement of postoperative therapy. Here we established a highly reproducible mouse GBM surgical model by using the syngeneic G422TN-GBM cells, which faithfully recapitulates the features of rapid recurrence of human GBM after surgery. Implanting 2 × 103-5 × 104 of G422TN-GBM cells in mouse cerebral cortex caused death in all animal within 23 days, while surgery was an effective therapy but not curable. After complete removal of visible tumors on day 5-9 of tumor growth, the tumors recurred macroscopically within 5 days accompanied by increasing infiltrative cancer foci. Mechanistically, the rapid recurrence of resected tumors was positively correlated to early Akt activation, which subsequently upregulated PD-L1/Vimentin and promoted proliferation/migration of cancer cells. In addition, environmental astrocytic activation with strong PD-L1 signal was prominent. Taken together, we provided a novel GBM surgical recurrence model for preclinical studies and suggested complicated recurring mechanisms involving in strong oncogenic signaling as well as immune inhibitory signals from both GBM cells and their neighboring astrocytes.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Vimentina/metabolismo , Animais , Neoplasias Encefálicas/cirurgia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Glioblastoma/cirurgia , Glioblastoma/terapia , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Recidiva Local de Neoplasia
10.
ACS Appl Mater Interfaces ; 13(21): 25044-25052, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34019375

RESUMO

Integration of novel bio-/nanostructures as effective sensing platforms is still of great significance for robust and rapid analysis. Herein, a novel metal-organic framework-derived NiCo2O4 was synthesized via a feasible templating method. Significantly, redox couples of both Ni3+/Ni2+ and Co3+/Co2+ provided richer oxidation-reduction reactions, thereby leading to an enhanced catalytic activity. Furthermore, NiCo2O4 as an enzyme mimic with peroxidase-like activity and oxidase-like activity could oxidize colorless thylbenzidine (TMB) to blue oxTMB in the absence of H2O2. Thus, a sensitive chromogenic sensing platform for detecting Fe2+, thiourea, cysteine (Cys), and epigallocatechin-3-gallate (EGCG) was proposed. The colorimetric detection methods exhibited great features of low limit of detection (LOD) and broad linear range. Owing to the complexation reaction, the chromogenic sensing system of TMB + NiCo2O4 + Cys achieved effective detection of Cu2+ and Mn2+ with the LODs of 0.0022 and 0.0181 mM, respectively. Developed detection methods with wide linear ranges of 0.008-0.1 mM for Cu2+ and 0.08-1 mM for Mn2+ had excellent practical potential. Similarly, the reaction system of TMB + NiCo2O4 + EGCG could achieve the colorimetric detection of Cu2+ and Fe3+. The great chromogenic sensing performance for detecting Cu2+ and Fe3+ with a broad linear range and a low LOD could be also realized.


Assuntos
Colorimetria/métodos , Enzimas/química , Estruturas Metalorgânicas/química , Metais/análise , Mimetismo Molecular , Catálise , Limite de Detecção , Oxirredução , Proteínas/química
11.
Elife ; 102021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33970104

RESUMO

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.


Assuntos
Liases de Carbono-Enxofre/química , Psychrobacter/enzimologia , Compostos de Sulfônio/metabolismo , Acil Coenzima A/metabolismo , Trifosfato de Adenosina , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Liases de Carbono-Enxofre/genética , Psychrobacter/genética , Psychrobacter/crescimento & desenvolvimento , Sulfetos/metabolismo
12.
Clin Neurol Neurosurg ; 205: 106651, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33940563

RESUMO

OBJECTIVES: This study aims to investigate the efficacy and safety of agomelatine, sertraline, and escitalopram for patients with senile post-stroke depression (SPSD, aged over 65 years). PATIENTS AND METHODS: A total of 165 patients (aged over 65 years) with post-stroke depression (PSD) were recruited. These patients were randomly assigned to one of four groups and given an anti-depressant or a placebo as follows: group A (agomelatine in combination with conventional cerebrovascular disease medication) 48 patients; group B (sertraline in combination with conventional cerebrovascular disease medication) 47 patients; group C (escitalopram in combination with conventional cerebrovascular disease medication) 50 patients; and, a control group (conventional treatment alone) 20 patients. The efficacy of the different treatments was evaluated using the Hamilton Depression Scale (HAMD), the National Institute of Health Stroke Scale (NIHSS), and the Activities of Daily Living (ADL) Barthel index (BI) at one, two, four, and six weeks after treatment began. RESULTS: According to the HAMD, NIHSS score, and BI index, the patients who received one of the three antidepressant treatments showed significant improvement compared with the control group (p < 0.05), but there was no significant difference between the three groups receiving anti-depressant medication (p > 0.05). Laboratory tests showed that the general adverse effects of the treatments were mild in all three groups, and patients generally tolerated the treatments. CONCLUSION: A decrease of HAMD and NIHSS scores and an increase in the BI index could be observed in the patients receiving agomelatine, sertraline, or escitalopram treatment. Thus, it would appear that the condition of SPSD in older patients can be improved with the use of either agomelatine, sertraline, or escitalopram.

13.
J Biol Chem ; 297(1): 100841, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34058201

RESUMO

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.


Assuntos
Acetilesterase/química , Acetilesterase/metabolismo , Adaptação Fisiológica , Temperatura Baixa , Acetilesterase/antagonistas & inibidores , Acetilesterase/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Cinética , Metais/farmacologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação/genética , Filogenia , Multimerização Proteica , Especificidade por Substrato/efeitos dos fármacos , Temperatura
14.
Antonie Van Leeuwenhoek ; 114(7): 947-955, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33864544

RESUMO

A novel Gram-negative, rod-shaped, aerobic, oxidase-positive and catalase-negative bacterium, designated strain SM1970T, was isolated from a seawater sample collected from the Mariana Trench. Strain SM1970T grew at 15-37 oC and with 1-5% (w/v) NaCl. It hydrolyzed colloidal chitin, agar and casein but did not reduce nitrate to nitrite. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SM1970T formed a distinct lineage close to the genus Catenovulum within the family Alteromonadaceae, sharing the highest sequence similarity (93.6%) with type strain of Catenovulum maritimum but < 93.0% sequence similarity with those of other known species in the class Gammaproteobacteria. The major fatty acids of strain SM1970T were summed feature 3 (C16: 1 ω7c and/or C16: 1 ω6c), C16: 0 and summed feature 8 (C18: 1 ω7c and/or C18: 1 ω6c). The major polar lipids of the strain included phosphatidylethanolamine and phosphatidylglycerol and its main respiratory quinone was ubiquinone 8. The draft genome of strain SM1970T consisted of 77 scaffolds and was 4,172,146 bp in length, containing a complete set of genes for chitin degradation. The average amino acid identity (AAI) values between SM1970T and type strains of known Catenovulum species were 56.6-57.1% while the percentage of conserved proteins (POCP) values between them were 28.5-31.5%. The genomic DNA G + C content of strain SM1970T was 40.1 mol%. On the basis of the polyphasic analysis, strain SM1970T is considered to represent a novel species in a novel genus of the family Alteromonadaceae, for which the name Marinifaba aquimaris is proposed with the type strain being SM1970T (= MCCC 1K04323T = KCTC 72844T).


Assuntos
Alteromonadaceae , Quitina , Alteromonadaceae/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Água do Mar , Análise de Sequência de DNA
15.
Appl Environ Microbiol ; 87(12): e0041221, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771786

RESUMO

Ulvan is an important marine polysaccharide. Bacterial ulvan lyases play important roles in ulvan degradation and marine carbon cycling. Until now, only a small number of ulvan lyases have been characterized. Here, a new ulvan lyase, Uly1, belonging to polysaccharide lyase family 24 (PL24) from the marine bacterium Catenovulum maritimum, is characterized. The optimal temperature and pH for Uly1 to degrade ulvan are 40°C and pH 9.0, respectively. Uly1 degrades ulvan polysaccharides in the endolytic manner, mainly producing ΔRha3S, consisting of an unsaturated 4-deoxy-l-threo-hex-4-enopyranosiduronic acid and a 3-O-sulfated α-l-rhamnose. The structure of Uly1 was resolved at a 2.10-Å resolution. Uly1 adopts a seven-bladed ß-propeller architecture. Structural and site-directed mutagenesis analyses indicate that four highly conserved residues, H128, H149, Y223, and R239, are essential for catalysis. H128 functions as both the catalytic acid and base, H149 and R239 function as the neutralizers, and Y223 plays a supporting role in catalysis. Structural comparison and sequence alignment suggest that Uly1 and many other PL24 enzymes may directly bind the substrate near the catalytic residues for catalysis, different from the PL24 ulvan lyase LOR_107, which adopts a two-stage substrate binding process. This study provides new insights into ulvan lyases and ulvan degradation. IMPORTANCE Ulvan is a major cell wall component of green algae of the genus Ulva. Many marine heterotrophic bacteria can produce extracellular ulvan lyases to degrade ulvan for a carbon nutrient. In addition, ulvan has a range of physiological bioactivities based on its specific chemical structure. Ulvan lyase thus plays an important role in marine carbon cycling and has great potential in biotechnological applications. However, only a small number of ulvan lyases have been characterized over the past 10 years. Here, based on biochemical and structural analyses, a new ulvan lyase of polysaccharide lyase family 24 is characterized, and its substrate recognition and catalytic mechanisms are revealed. Moreover, a new substrate binding process adopted by PL24 ulvan lyases is proposed. This study offers a better understanding of bacterial ulvan lyases and is helpful for studying the application potentials of ulvan lyases.


Assuntos
Alteromonadaceae/enzimologia , Polissacarídeo-Liases/química , Sequência de Aminoácidos , Catálise , Filogenia , Polissacarídeo-Liases/genética , Polissacarídeos/química , Especificidade por Substrato
16.
Sci Adv ; 7(13)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33771875

RESUMO

High hydrostatic pressure (HHP) is a characteristic environmental factor of the deep ocean. However, it remains unclear how piezotolerant bacteria adapt to HHP. Here, we identify a two-step metabolic pathway to cope with HHP stress in a piezotolerant bacterium. Myroides profundi D25T, obtained from a deep-sea sediment, can take up trimethylamine (TMA) through a previously unidentified TMA transporter, TmaT, and oxidize intracellular TMA into trimethylamine N-oxide (TMAO) by a TMA monooxygenase, MpTmm. The produced TMAO is accumulated in the cell, functioning as a piezolyte, improving both growth and survival at HHP. The function of the TmaT-MpTmm pathway was further confirmed by introducing it into Escherichia coli and Bacillus subtilis Encoded TmaT-like and MpTmm-like sequences extensively exist in marine metagenomes, and other marine Bacteroidetes bacteria containing genes encoding TmaT-like and MpTmm-like proteins also have improved HHP tolerance in the presence of TMA, implying the universality of this HHP tolerance strategy in marine Bacteroidetes.

18.
J Biol Chem ; 296: 100081, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33199371

RESUMO

Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles.


Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Glutamatos/metabolismo , Trifosfato de Adenosina/metabolismo , Catálise , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Magnésio/metabolismo , Metilaminas/metabolismo , Microscopia Eletrônica , Rhodovulum/metabolismo
19.
Cell Discov ; 6(1): 97, 2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33372178

RESUMO

Germ cells are vital for reproduction and heredity. However, the mechanisms underlying female germ cell development in primates, especially in late embryonic stages, remain elusive. Here, we performed single-cell RNA sequencing of 12,471 cells from whole fetal ovaries, and explored the communications between germ cells and niche cells. We depicted the two waves of oogenesis at single-cell resolution and demonstrated that progenitor theca cells exhibit similar characteristics to Leydig cells in fetal monkey ovaries. Notably, we found that ZGLP1 displays differentially expressed patterns between mouse and monkey, which is not overlapped with NANOG in monkey germ cells, suggesting its role in meiosis entry but not in activating oogenic program in primates. Furthermore, the majority of germ cell clusters that sharply express PRDM9 and SPO11 might undergo apoptosis after cyst breakdown, leading to germ cell attrition. Overall, our work provides new insights into the molecular and cellular basis of primate fetal ovary development at single-cell resolution.

20.
Food Sci Nutr ; 8(10): 5738-5747, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33133575

RESUMO

Phthalate acid esters (PAEs) are one of the essential plastic additives which may lead to plenty of harmful effects, including reproductive toxicity, teratogenicity, and carcinogenicity. Increasing attention has been paid to the migration of plasticizer. In this article, the disposable plastic lunch boxes were taken as the research object. The result showed that dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) have been mainly found, whose content was 1.5 mg/kg and 2.4 mg/kg, respectively. The LOD was 2 ng/g, and LOQ was 6.7 ng/g. We further investigated the migration of PAEs into the simulated liquid at different temperature conditions. Then, the linear fitting performing by first-order kinetic migration model revealed that the lower the polarity of the simulated liquid, the larger the rate constant K 1 and initial release rate V 0. The higher the temperature, the bigger the K 1 and V 0.

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