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1.
Mar Drugs ; 19(12)2021 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-34940675

RESUMO

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme-substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.

2.
Environ Microbiol ; 2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34913576

RESUMO

Based on 16S rRNA gene analyses, the same bacterial operational taxonomic units (OTUs) are common to both the Arctic and Antarctic oceans, supporting the concept 'everything is everywhere'. However, whether the same OTUs from both poles have identical genomes, i.e. whether 'everything is still everywhere' at the genomic level has not yet been examined systematically. Here, we isolated, sequenced and compared the genomes of 45 culturable marine bacteria belonging to three genera of Salinibacterium, Psychrobacter and Pseudoalteromonas from both polar oceans. The bacterial strains with identical 16S rRNA genes were common to both poles in every genus, and four identical genomes were detected in the genus Salinibacterium from the Arctic region. However, no identical genomes were observed from opposite poles in this study. Our data, therefore, suggest that 'everything is not everywhere' at the genomic level. The divergence time between bacteria is hypothesized to exert a strong impact on the bacterial biogeography at the genomic level. The geographical isolation between poles was observed for recently diverged, highly similar genomes, but not for moderately similar genomes. This study thus improves our understanding of the factors affecting the genomic-level biogeography of marine microorganisms isolated from distant locations.

3.
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.

4.
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.

6.
Front Microbiol ; 12: 721422, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34659148

RESUMO

1,3-xylan is present in the cell walls of some red and green algae and is an important organic carbon in the ocean. However, information on its bacterial degradation is quite limited. Here, after enrichment with 1,3-xylan, the diversity of bacteria recovered from marine algae collected in Hainan, China, was analyzed with both the 16S rRNA gene amplicon sequencing and the culture-dependent method. Bacteria recovered were affiliated with more than 19 families mainly in phyla Proteobacteria and Bacteroidetes, suggesting a high bacterial diversity. Moreover, 12 strains with high 1,3-xylanase-secreting ability from genera Vibrio, Neiella, Alteromonas, and Gilvimarinus were isolated from the enrichment culture. The extracellular 1,3-xylanases secreted by Vibrio sp. EA2, Neiella sp. GA3, Alteromonas sp. CA13-2, and Gilvimarinus sp. HA3-2, which were taken as representatives due to their efficient utilization of 1,3-xylan for growth, were further characterized. The extracellular 1,3-xylanases secreted by these strains showed the highest activity at pH 6.0-7.0 and 30-40°C in 0-0.5M NaCl, exhibiting thermo-unstable and alkali-resistant characters. Their degradation products on 1,3-xylan were mainly 1,3-xylobiose and 1,3-xylotriose. This study reveals the diversity of marine bacteria involved in the degradation and utilization of 1,3-xylan, helpful in our understanding of the recycling of 1,3-xylan driven by bacteria in the ocean and the discovery of novel 1,3-xylanases.

7.
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.

8.
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
9.
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.

10.
Appl Environ Microbiol ; 87(22): e0116021, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34469192

RESUMO

Nordic Seas are the subarctic seas connecting the Arctic Ocean and North Atlantic Ocean with complex water masses, experiencing an abrupt climate change. Though knowledge of the marine virosphere has expanded rapidly, the diversity of viruses and their relationships with host cells and water masses in the Nordic Seas remain to be fully revealed. Here, we establish the Nordic Sea DNA virome (NSV) data set of 55,315 viral contigs including 1,478 unique viral populations from seven stations influenced by both the warm Atlantic and cold Arctic water masses. Caudovirales dominated in the seven NSVs, especially in the warm Atlantic waters. The major giant nucleocytoplasmic large DNA viruses (NCLDVs) contributed a significant proportion of the classified viral contigs in the NSVs (32.2%), especially in the cold Arctic waters (44.9%). The distribution patterns of Caudovirales and NCLDVs were a reflection of the community structure of their hosts in the corresponding water masses and currents. Latitude, pH, and flow speed were found to be key factors influencing the microbial communities and coinfluencing the variation of viral communities. Network analysis illustrated the tight coupling between the variation of viral communities and microbial communities in the Nordic Seas. This study suggests a probable linkage between viromes, host cells, and surface water masses from both the cool Arctic and warm Atlantic Oceans. IMPORTANCE This is a systematic study of Nordic Sea viromes using metagenomic analysis. The viral diversity, community structure, and their relationships with host cells and the complex water masses from both the cool Arctic and the warm Atlantic oceans were illustrated. The NCLDVs and Caudovirales are proposed as the viral characteristics of the cold Arctic and warm Atlantic waters, respectively. This study provides an important background for the viromes in the subarctic seas connecting the Arctic Ocean and North Atlantic Ocean and sheds light on their responses to abrupt climate change in the future.

11.
Mar Genomics ; 59: 100874, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34493388

RESUMO

Pelagovum pacificum SM1903T, belonging to a novel genus of the family Rhodobacteraceae, was isolated from the surface seawater of the Mariana Trench. Here, we report the first complete genome sequence of the novel genus Pelagovum. The genome of strain SM1903T consists of a circular chromosome of 4,040,866 bp and two plasmids of 41,363 bp and 9705 bp, respectively. Gene annotation and metabolic pathway analyses showed that strain SM1903T possesses a series of genes related to adaptation to marine oligotrophic environments, which are involved in utilization of aromatic compounds, allantoin, and alkylphosphonate, and second messenger signaling in response to the oligotrophic stress. This strain also contains a variety of genes involved in coping with other stresses including osmotic stress, oxidative stress, cold shock, and heat shock. These features would assist this strain to survive under the natural nutrient limitation and other stresses from the environment. The genome of strain SM1903T of the novel genus Pelagovum would deepen our knowledge on marine bacterioplankton and their adaption strategies to marine oligotrophic environments.


Assuntos
Genoma Bacteriano , Rhodobacteraceae , Composição de Bases , Filogenia , Rhodobacteraceae/genética , Água do Mar
12.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361020

RESUMO

A stochastic model of the flow cytometer measurement process was developed to assess the nature of the observed coefficient of variation (CV%) of the mean fluorescence intensity (MFI) from a population of labeled microspheres (beads). Several sources of variability were considered: the total number of labels on a bead, the path through the laser beam, the optical absorption cross-section, the quantum yield, the numerical aperture of the collection optics, and the photoelectron conversion efficiency of the photomultiplier (PMT) cathode. The variation in the number of labels on a bead had the largest effect on the CV% of the MFI of the bead population. The variation in the path of the bead through the laser beam was minimized using flat-top lasers. The variability in the average optical properties of the labels was of minor importance for beads with sufficiently large number of labels. The application of the bead results to the measured CV% of labeled B cells indicated that the measured CV% was a reliable measure of the variability of antibodies bound per cell. With some modifications, the model can be extended to multicolor flow cytometers and to the study of CV% from cells with low fluorescence signal.


Assuntos
Linfócitos B/citologia , Citometria de Fluxo/normas , Microesferas , Análise de Variância , Citometria de Fluxo/métodos , Humanos , Reprodutibilidade dos Testes , Processos Estocásticos
13.
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.

14.
Curr Med Sci ; 41(4): 695-704, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34403094

RESUMO

OBJECTIVE: Auditory neuropathy (AN) is a unique pattern of hearing loss with preservation of hair cell function. The condition is characterized by the presence of otoacoustic emissions (OAE) or cochlear microphonic (CM) responses with severe abnormalities of the auditory brainstem response (ABR). The vestibular branches of the VIII cranial nerve and the structures innervated by it can also be affected. However, the precise lesion sites in the vestibular system are not well characterized in patients with AN. METHODS: The air-conducted sound (ACS) vestibular-evoked myogenic potentials (VEMPs) and galvanic vestibular stimuli (GVS)-VEMPs were examined in 14 patients with AN. RESULTS: On examination of VEMPs (n=14, 28 ears), the absent rates of ACS-cervical VEMP (cVEMP), ACS-ocular VEMP (oVEMP), GVS-cVEMP, GVS-oVEMP and caloric test were 92.9% (26/28), 85.7% (24/28), 67.9% (19/28), 53.6% (15/28), and 61.5% (8/13), respectively. Impaired functions of the saccule, inferior vestibular nerve, utricle, superior vestibular nerve, and horizontal semicircular canal were found in 25.0% (7/28), 67.9% (19/28), 32.1% (9/28), 53.6% (15/28) and 61.5% (8/13) patients, respectively. On comparing the elicited VEMPs parameters of AN patients with those of normal controls, both ACS-VEMPs and GVS-VEMPs showed abnormal results in AN patients (such as, lower presence rates, elevated thresholds, prolonged latencies, and decreased amplitudes). CONCLUSION: The study suggested that patients with AN often have concomitant vestibular disorders. Retro-labyrinthine lesions were more frequently observed in this study. GVS-VEMPs combined with ACS-VEMPs may help identify the lesion sites and facilitate detection of areas of vestibular dysfunction in these patients.

15.
Appl Environ Microbiol ; 87(17): e0036821, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34160244

RESUMO

Alginate, which is mainly produced by brown algae and decomposed by heterotrophic bacteria, is an important marine organic carbon source. The genus Pseudoalteromonas contains diverse forms of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation. In this review, the diversity of alginate-degrading Pseudoalteromonas is introduced, and the characteristics of Pseudoalteromonas alginate lyases, including their sequences, enzymatic properties, structures, and catalytic mechanisms, and the synergistic effect of Pseudoalteromonas alginate lyases on alginate degradation are introduced. The acquisition of the alginate degradation capacity and the alginate utilization pathways of Pseudoalteromonas are also introduced. This paper provides a comprehensive overview of alginate degradation by Pseudoalteromonas, which will contribute to the understanding of the degradation and recycling of marine algal polysaccharides driven by marine bacteria.


Assuntos
Alginatos/metabolismo , Pseudoalteromonas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feófitas/metabolismo , Feófitas/microbiologia , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Pseudoalteromonas/química , Pseudoalteromonas/enzimologia , Pseudoalteromonas/genética , Água do Mar/microbiologia
16.
Antonie Van Leeuwenhoek ; 114(8): 1205-1212, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33973093

RESUMO

The cell of a rod-shaped bacterium is composed of a cylinder and two hemispherical poles. In recent decades, the molecular mechanism of morphogenesis in rod-shaped bacteria has received extensive research. However, most works have focused on the morphogenesis of cylinders, and the morphogenesis of the hemispherical poles remains unclear. In the past, the pole of bacterial cell wall was considered as a rigid hemispherical structure. However, our work indicated that the pole in the isolated sacculi from Bacillus subtilis was a flat structure instead of a hemisphere form. Further works showed that internal pressure was responsible for shaping the hemispherical poles, indicating an elastic nature of the cell wall in poles. In addition, we found that the internal pressure was able to transform septa into hemispherical shape which is similar to normal poles. Based on our work, we proposed a model for the internal pressure-induced formation of hemispherical poles in B. subtilis, and this work may provide new clues into basic knowledge of the morphogenesis of rod-shaped bacteria.


Assuntos
Bacillus subtilis , Parede Celular , Bacillus subtilis/genética , Proteínas de Bactérias , Morfogênese
17.
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
18.
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
19.
FEBS J ; 288(19): 5768-5780, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33843134

RESUMO

Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal ß-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH'S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical α/ß-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand ß6. MpaH' also forms an atypical dimer with the unique C-terminal helices α13 and α14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of α/ß hydrolase fold protein. The crystal structure of MpaH'S139A /MPA complex and the modeled structure of MpaH'/MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.


Assuntos
Acil Coenzima A/química , Hidrolases/ultraestrutura , Ácido Micofenólico/metabolismo , Peroxissomos/ultraestrutura , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Hidrolases/química , Hidrolases/genética , Ácido Micofenólico/química , Penicillium/genética , Penicillium/ultraestrutura , Peroxissomos/enzimologia , Estrutura Secundária de Proteína/genética , Especificidade por Substrato/genética
20.
Front Microbiol ; 12: 658620, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815349

RESUMO

Deconstruction is an essential step of conversion of polysaccharides, and polysaccharide-degrading enzymes play a key role in this process. Although there is recent progress in the identification of these enzymes, the diversity and phylogenetic distribution of these enzymes in marine microorganisms remain largely unknown, hindering our understanding of the ecological roles of marine microorganisms in the ocean carbon cycle. Here, we studied the phylogenetic distribution of nine types of polysaccharide-degrading enzymes in marine bacterial genomes. First, we manually compiled a reference sequence database containing 961 experimentally verified enzymes. With this reference database, we annotated 9,335 enzyme sequences from 2,182 high-quality marine bacterial genomes, revealing extended distribution for six enzymes at the phylum level and for all nine enzymes at lower taxonomic levels. Next, phylogenetic analyses revealed intra-clade diversity in the encoding potentials and phylogenetic conservation of a few enzymes at the genus level. Lastly, our analyses revealed correlations between enzymes, with alginate lyases demonstrating the most extensive correlations with others. Intriguingly, chitinases showed negative correlations with cellulases, alginate lyases, and agarases in a few genera. This result suggested that intra-genus lifestyle differentiation occurred many times in marine bacteria and that the utilization of polysaccharides may act as an important driver in the recent ecological differentiation of a few lineages. This study expanded our knowledge of the phylogenetic distribution of polysaccharide enzymes and provided insights into the ecological differentiation of marine bacteria.

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