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Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s−1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

It has been a long time since the first α-agarase was discovered. However, only two α-agarases have been cloned and partially characterized so far and the study of α-agarases has lagged far behind that of β-agarases. Here, we report an α-agarase, AgaD, cloned from marine bacterium Thalassomonas sp. LD5. Its cDNA consists of 4401 bp, encoding a protein of 1466 amino acids. Based on amino acid similarity, AgaD is classified into glycoside hydrolase (GH) family GH96. The recombinant enzyme gave a molecular weight of about 180 kDa on SDS-PAGE and 360 kDa on Native-PAGE indicating it acted as a dimer. However, the recombinant enzyme is labile and easy to be fractured into series of small active fragments, of which the smallest one is about 70 kDa, matching the size of catalytic module. The enzyme has maximal activity at 35 °C and pH 7.4, and shows a strong dependence on the presence of calcium ions. AgaD degrades agarose to yield agarotetraose as the predominate end product. However, the hydrolysates are rapidly degraded to odd-numbered oligosaccharides under strong alkaline condition. The spectra of ESI-MS and 1H-NMR proved that the main hydrolysate agarotetraose is degraded into neoagarotriose, bearing the sequence of G-A-G (G, d-galactose; A, 3,6-anhydro-α-l-galactose). Unlike the alkaline condition, the hydrolysates are further hydrolyzed into smaller degree polymerization (DP) of agaro-oligosaccharides (AOS) in dilute strong acid. Therefore, this study provides more insights into the properties for both the α-agarases and the AOS.

Marine biofilms are a collective of microbes that can grow on many different surfaces immersed in marine environments. Estimating the microbial richness and specificity of a marine biofilm community is a challenging task due to the high complexity in comparison with seawater. Here, we compared the resolution of full-length 16S rRNA gene sequencing technique of a PacBio platform for microbe identification in marine biofilms with the results of partial 16S rRNA gene sequencing of traditional Illumina PE250 platform. At the same time, the microbial richness, diversity, and composition of adjacent seawater communities in the same batch of samples were analyzed. Both techniques revealed higher species richness, as reflected by the Chao1 index, in the biofilms than that in the seawater communities. Moreover, compared with Illumina sequencing, PacBio sequencing detected more specific species for biofilms and less specific species for seawater. Members of Vibrio, Arcobacter, Photobacterium, Pseudoalteromonas, and Thalassomonas were significantly enriched in the biofilms, which is consistent with the previous understanding of species adapted to a surface-associated lifestyle and validates the taxonomic analyses in the current study. To conclude, the full-length sequencing of 16S rRNA genes has probably a stronger ability to analyze more complex microbial communities, such as marine biofilms, the species richness of which has probably been under-estimated in previous studies.

Although many β-agarases that hydrolyze the β-1,4 linkages of agarose have been biochemically characterized, only three α-agarases that hydrolyze the α-1,3 linkages are reported to date. In this study, a new α-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an α-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca 2+ , and was strongly inhibited by EDTA. The K m and V max of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type α-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing α-agarase was identified.

Alginate oligosaccharides (AOS) and their derivatives become popular due to their favorable biological activity, and the key to producing functional AOS is to find efficient alginate lyases. This study showed one alginate lyase TsAly7A found in Thalassomonas sp. LD5, which was predicted to have excellent industrial properties. Bioinformatics analysis and enzymatic properties of recombinant TsAly7A (rTsAly7A) were investigated. TsAly7A belonged to the fifth subfamily of polysaccharide lyase family 7 (PL7). The optimal temperature and pH of rTsAly7A was 30 °C and 9.1 in Glycine-NaOH buffer, respectively. The pH stability of rTsAly7A under alkaline conditions was pretty good and it can remain at above 90% of the initial activity at pH 8.9 in Glycine-NaOH buffer for 12 h. In the presence of 100 mM NaCl, rTsAly7A showed the highest activity, while in the absence of NaCl, 50% of the highest activity was observed. The rTsAly7A was an endo-type alginate lyase, and its end-products of alginate degradation were unsaturated oligosaccharides (degree of polymerization 2–6). Collectively, the rTsAly7A may be a good industrial production tool for producing AOS with high degree of polymerization.

Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment.

The coastal environments worldwide are subjected to increasing TBBPA contamination, but current knowledge on aerobic biodegradability of this compound by marine microbes is lacking. The aerobic removal of TBBPA using marine consortia under eight different cometabolic conditions was investigated here. Results showed that the composition and diversity of the TBBPA-degrading consortia had diverged after 120-day incubation. Pseudoalteromonas , Alteromonas , Glaciecola , Thalassomonas , and Limnobacter were the dominant genera in enrichment cultures. Furthermore, a combination of beef extract- and peptone-enriched marine consortia exhibited higher TBBPA removal efficiency (approximately 60%) than the other substrate amendments. Additionally, Alteromonas macleodii strain GCW was isolated from a culture of TBBPA-degrading consortium. This strain exhibited about 90% of degradation efficiency toward TBBPA (10 mg L −1 ) after 10 days of incubation under aerobic cometabolic conditions. The intermediates in the degradation of TBBPA by A. macleodii strain GCW were analyzed and the degradation pathways were proposed, involving β -scission, debromination, and nitration routes.

A Gram-negative, aerobic, non-motile, dark brown-coloured and rod-shaped bacterial strain, designated G-MB1 T , was isolated from a tidal flat sediment of the South Sea, South Korea. Strain G-MB1 T was found to grow optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain G-MB1 T fell within the clade comprising Thalassomonas species, clustering with the type strains of Thalassomonas agarivorans , Thalassomonas loyana , Thalassomonas ganghwensis and Thalassomonas agariperforans , with which it exhibited 16S rRNA gene sequence similarity values of 96.0–96.9 %. The 16S rRNA gene sequence similarity values between strain G-MB1 T and the type strains of the other Thalassomonas species were 94.6–95.1 %. Strain G-MB1 T was found to contain Q-8 as the predominant ubiquinone and C 16:0 , C 17:1 ω 8 c , C 16:1 ω 9 c , C 12:0 3-OH and summed feature 3 (C 16:1 ω 7 c and/or C 16:1 ω 6 c ) as the major fatty acids. The major polar lipids of strain G-MB1 T were phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid. The DNA G+C content of strain G-MB1 T was determined to be 42.4 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain G-MB1 T is separated from other Thalassomonas species. On the basis of the data presented, strain G-MB1 T is considered to represent a novel species of the genus Thalassomonas , for which the name Thalassomonas fusca sp. nov. is proposed. The type strain is G-MB1 T (=KCTC 32499 T  = NBRC 109830 T ).

The Brazilian endemic scleractinian corals, genus Mussismilia, are among the main reef builders of the South Atlantic and are threatened by accelerating rates of disease. To better understand how holobiont microbial populations interact with corals during health and disease and to evaluate whether selective pressures in the holobiont or neutral assembly shape microbial composition, we have examined the microbiota structure of Mussismilia corals according to coral lineage, environment, and disease/health status. Microbiota of three Mussismilia species (Mussismilia harttii, Mussismilia hispida, and Mussismilia braziliensis) was compared using 16S rRNA pyrosequencing and clone library analysis of coral fragments. Analysis of biological triplicates per Mussismilia species and reef site allowed assessment of variability among Mussismilia species and between sites for M. braziliensis. From 173,487 V6 sequences, 6,733 coral- and 1,052 water-associated operational taxonomic units (OTUs) were observed. M. braziliensis microbiota was more similar across reefs than to other Mussismilia species microbiota from the same reef. Highly prevalent OTUs were more significantly structured by coral lineage and were enriched in Alpha- and Gammaproteobacteria. Bacterial OTUs from healthy corals were recovered from a M. braziliensis skeleton sample at twice the frequency of recovery from water or a diseased coral suggesting the skeleton is a significant habitat for microbial populations in the holobiont. Diseased corals were enriched with pathogens and opportunists (Vibrios, Bacteroidetes, Thalassomonas, and SRB). Our study examines for the first time intra- and inter-specific variability of microbiota across the genus Mussismilia. Changes in microbiota may be useful indicators of coral health and thus be a valuable tool for coral reef management and conservation.