Agarivorans Albus Research Articles

Direct Degradation of Fresh and Dried Macroalgae by Agarivorans albus B2Z047.

Marine macroalgae are increasingly recognized for their significant biological and economic potential. The key to unlocking this potential lies in the efficient degradation of all carbohydrates from the macroalgae biomass. However, a variety of polysaccharides (alginate, cellulose, fucoidan, and laminarin), are difficult to degrade simultaneously in a short time. In this study, the brown alga Saccharina japonica was found to be rapidly and thoroughly degraded by the marine bacterium Agarivorans albus B2Z047. This strain harbors a broad spectrum of carbohydrate-active enzymes capable of degrading various polysaccharides, making it uniquely equipped to efficiently break down both fresh and dried kelp, achieving a hydrolysis rate of up to 52%. A transcriptomic analysis elucidated the presence of pivotal enzyme genes implicated in the degradation pathways of alginate, cellulose, fucoidan, and laminarin. This discovery highlights the bacterium's capability for the efficient and comprehensive conversion of kelp biomass, indicating its significant potential in biotechnological applications for macroalgae resource utilization.

Isolation and pathogenicity identification of bacterial pathogens in bleached disease and their physiological effects on the red macroalga Gracilaria lemaneiformis

Marine macroalga, Gracilaria lemaneiformis, is affected by bleached disease in recent years, but the bacterial pathogens for bleached disease in G. lemaneiformis are either known or unknown. In this study, we identified eight bacterial pathogens, of which Agarivorans albus, Aquimarina latercula (T) and Brachybacterium sp. had the strongest pathogenicity on healthy G. lemaneiformis. These pathogens had strong agarase activity, suggesting a possible infection mechanism through the degradation of algal-agar. Furthermore, analysis of the macroalgal defense against pathogens in terms of hydrogen peroxide and malondialdehyde content indicated that oxidative burst started at an early stage during the bacterial infection. On the other hand, the increasing activity of peroxidase and the occurrence of different peroxidases were to protect algae from the oxidative damage, by compensating for the decrease in catalase and total antioxidant capacity in bleached tissue. The photosynthetic pigment – phycobiliprotein was dramatically decreased after the bacterial infection, with the beta subunit but not the alpha subunit of phycoerythrin being degraded first as the disease progressed. The findings here provide very important fundamental information for future research on understanding the algae-bacteria interaction mechanism, the mechanism of algal defense against bacterial infection, which is important for the efficient and healthy cultivation of G. lemaneiformis.

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli

ABSTRACTAgarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 9 h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ∼10 mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu2+, Mn2+, Zn2+, and Al3+ ions. Furthermore, the KM and kcat values for purified Aaβ-agarase were ∼0.02 mM and ∼45/s, respectively. These kinetic values were up to approximately 15–100-fold lower than the KM values reported for other agarases and approximately 7–30-fold higher than the kcat/KM values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.

Agarivorans aestuarii sp. nov., an agar-degrading bacterium isolated from a tidal flat.

A Gram-reaction-negative, aerobic, non-spore forming, rod-shaped bacterium motile with a single polar flagellum, designated strain hydD622T, was isolated from the sediment of a tidal flat at Asan Bay, Korea. Strain hydD622T exhibited an agarolytic activity. Comparison of 16S rRNA gene sequences revealed that strain hydD622T was closely related to Agarivorans litoreus KCTC 42116T, Agarivorans albus KCTC 22256T and Agarivorans gilvus KCTC 32555T with similarities of 98.4, 98.0 and 96.5 %, respectively. Strain hydD622T was clustered distantly from the other genera in the family Alteromonadaceae but formed a unique clade within the genus Agarivorans based on the 16S rRNA gene sequence. The DNA-DNA relatedness with Agarivorans litoreus KCTC 42116T and Agarivorans. albus KCTC 22256T was 39.0 and 37.8 %, respectively. The major fatty acids (>10 %) were C16 : 0,C16 : 1ω6c/C16 : 1ω7c and C18 : 1ω6c/C18 : 1ω7c. The respiratory quinone was ubiquinone-8, and the polar lipid profile consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unidentified lipid. The DNA G+C content was 44 mol%. On the basis of physiological, chemotaxonomic and phylogenetic analyses, strain hydD622T represents a novel species within the genus Agarivorans, for which the name Agarivorans aestuarii sp. nov. is proposed. The type strain of Agarivorans aestuarii sp. nov. is hydD622T (=KCTC 32543T=CGMCC 1.12692T).

Expression and characterization of a thermostable and pH-stable β-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1

A novel β-agarase gene aga4383 was cloned from Flammeovirga pacifica WPAGA1. The gene consists of 2898 bp and encodes a protein, designated as AgaP4383, with 965 amino acids. The DNA sequence of aga4383 has no significant sequence similarity with any known proteins, including all glycoside hydrolases. AgaP4383 shares a highest amino acid sequence homology of 41% with a putative β-agarase from Agarivorans albus. Phylogenetic analysis showed that AgaP4383 belongs to family 86 of glycoside hydrolases (GH86). The agarase gene was expressed in Escherichia coli and purified by affinity chromatography. The purified AgaP4383 showed endolytic activity on agar degradation, yielding neoagarotetraose and neoagarohexaose as the end products. The Km values for agar and Gracilaria lemaneiformis were 8.53 and 32.41mgmL−1. The optimal temperature and pH for the recombinant AgaP4383 were 50°C and pH 9.0, respectively. Notably, the enzyme exhibited good thermostability. No activity loss was observed after incubation at 50°C for 10h. The recombinant AgaP4383 showed a wide range of pH stability, retaining 90% of activity at pH 5.0–10.0 for 24h at 30°C. These beneficial characteristics of the enzyme provide some advantages for potential application in industry.

Agarivorans litoreus sp. nov., a novel gammaproteobacterium isolated from seawater and emended description of the genus Agarivorans.

A Gram-stain negative, aerobic, non-spore-forming, motile and rod-shaped or ovoid bacterial strain, designated GJSW-6(T), was isolated from a seawater collected at Geoje island on the South Sea, South Korea, and subjected to polyphasic taxonomic study. Strain GJSW-6(T) was found to grow optimally at 30°C, at pH 7.0-8.0 and in the presence of approximately 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GJSW-6(T) clustered with the type strains of two Agarivorans species, Agarivorans albus and Agarivorans gilvus, with which it exhibited sequence similarity values of 99.17 and 95.88%, respectively. Sequence similarities to the type strains of the other recognized species were less than 93.04%. Strain GJSW-6(T) was found to contain Q-8 as the predominant ubiquinone and C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and C18:1 ω7c as the major fatty acids. The polar lipid profile of strain GJSW-6(T) containing phosphatidylethanolamine and phosphatidylglycerol as major components was similar to those of the type strains of A. albus and A. gilvus. The DNA G+C content of strain GJSW-6(T) was determined to be 45.3mol% and its mean DNA-DNA relatedness value with A. albus JCM 21469(T) was 19%. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain GJSW-6(T) is separated from two Agarivorans species. On the basis of the data presented, strain GJSW-6(T) is considered to represent a novel species of the genus Agarivorans, for which the name Agarivorans litoreus sp. nov. is proposed. The type strain is GJSW-6(T) (=KCTC 42116(T)=NBRC 110444(T)). An emended description of the genus Agarivorans is also proposed.

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06U/ml to 2.65U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338U/mg, respectively. The molecular masses of the agarases were estimated to be 50kDa and 107kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40°C and 50°C, respectively. Agarase-a was stable at 30°C, while agarase-b was stable at 50°C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu2+, Mn2+, Co2+ and EDTA, while agarase-b was inhibited by Cu2+, Mn2+ and Fe2+. The results of 13C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.

Draft Genome Sequence of Agarivorans albus Strain MKT 106 T , an Agarolytic Marine Bacterium

Agarivorans albus is a Gram-negative, strictly aerobic, and agar-hydrolyzing marine bacterium. We present the draft genome sequence of the A. albus strain MKT 106T, which is composed of 67 contigs (>500 bp) totaling 4,734,285 bp and containing 4,397 coding DNA sequences (CDSs), four rRNAs, and 64 tRNA sequences.

Identification of a marine agarolytic bacterium Agarivorans albus QM38 and cloning and sequencing its beta-agarase genes

A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negative, strictly aerobic, curved rod and polar flagellum. On the basis of several phenotypic characters, biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA, the strain was identified as Agarivorans albus strain QM38. This strain can liquefy the agar on the solid agar plate. An excellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40 °C, pH 7.6. Its activity was greatly affected by different concentrations of agarose. The highest activity 32 U/ml was achieved in the culture supernatant. The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). After complete hydrolysis of agarose, a series of agaro-oligosaccharides were produced. The main products of the enzymes were oligosaccharides in the degree of polymerization (DP) of 2, 4, 6 and 8. Three genes agaD01, agaD02 and agaD03, encoding β-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaD01, consisted of 2 988 bp, and shared 95.5%–98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans. Gene agaD02 comprised 2 868 bp and encoded a 955- amino-acid protein. It showed 97.4% and 98.7% identity to the β-agarase genes of strain Vibrio sp. PO-303 and strain Vibrio sp. JT0107, respectively. Only partial sequence of agaD03 gene has been cloned. It showed 96.5% identity to β-agarase gene (agaB) of Pseudoalteromonas sp. CY24, and shared 96.8% identity to β-agarase-c gene of Vibrio sp. PO-303.

Use of macroalgae supplemented with probiotics in the Haliotis rufescens (Swainson, 1822) culture in Northern Chile

In this study, we evaluate the use of macroalgae as vectors of probiotics bacteria into the digestive tract of abalone to improve their survival and growth. It is shown that when abalone Haliotis rufescens of different sizes were fed with a natural diet composed of fronds of the macroalga Macrocystis integrifolia supplemented with a mixture of Vibrio sp. C21-UMA, Agarivorans albus F1-UMA and Vibrio sp. F15-UMA bacteria, there was a significant increase (P<0.05) in the average monthly growth rate and survival (%)in a period of 210 days, compared with the control without a probiotic supplement. The permanence of the probiotics in the digestive tract of the animals was monitored, and it was found that the number of culturable C21-UMA and F1-UMA bacteria decreased significantly (P<0.05) in recently weaned and adult abalone, and they almost disappeared completely on day 19 of the bioassay. However, the culturable Vibrio sp. F15-UMA disappeared completely from the digestive tract on day 22 of the bioassay, and these were the bacteria that remained at the highest concentration compared with the other two bacterial strains over the experimental period. It is therefore shown that it is feasible to use a probiotic mixture to improve the profitability of the H. rufescens culture.

대장균에서 단백질 분비에 대한 Agarivorans albus YKW-34의 Agarase 시그널펩티드의 효과

To overcome the limitation of E. coli expression system such as inclusion body formation and disulfide bond failure, we tried to express the heterologous protein as a secreted form. We adopted agarase signal peptide (ASP; 23 amino acid residues) from Agarivorans albus YKW-34 which is one of marine bacteia. When we used ASP to express <TEX>$\beta$</TEX>-agarase, about 42% activity was detected in media.

Aliagarivorans marinus gen. nov., sp. nov. and Aliagarivorans taiwanensis sp. nov., facultatively anaerobic marine bacteria capable of agar degradation

Two agarolytic strains of Gram-negative, heterotrophic, facultatively anaerobic, marine bacteria, designated AAM1T and AAT1T, were isolated from seawater samples collected in the shallow coastal region of An-Ping Harbour, Tainan, Taiwan. Cells grown in broth cultures were straight rods that were motile by means of a single polar flagellum. The two isolates required NaCl for growth and grew optimally at about 25-30 degrees C, in 2-4% NaCl and at pH 8. They grew aerobically and could achieve anaerobic growth by fermenting D-glucose or other sugars. The major isoprenoid quinone was Q-8 (79.8-92.0%) and the major cellular fatty acids were summed feature 3 (C16:1omega7c and/or iso-C15:0 2-OH; 26.4-35.6%), C18:1omega7c (27.1-31.4%) and C16:0 (14.8-16.3%) in the two strains. Strains AAM1T and AAT1T had DNA G+C contents of 52.9 and 52.4 mol%, respectively. The two strains had a 16S rRNA gene sequence similarity of 98.6% and shared 84.9-92.4% sequence similarity with the type strains of Agarivorans albus (91.2-92.4%), eight Alteromonas species (84.9-87.1%), two Aestuariibacter species (86.0-87.0%), Bowmanella denitrificans (86.1-86.7%), eight Glaciecola species (85.0-87.9%) and Salinimonas chungwhensis (85.9-86.1%). Despite their high sequence similarity, strains AAM1T and AAT1T had a DNA-DNA relatedness value of only 4.5%. The data obtained from these polyphasic taxonomic studies revealed that the two agarolytic isolates could be classified as representatives of two novel species in a new genus, Aliagarivorans gen. nov., with Aliagarivorans marinus sp. nov. [type strain is AAM1T (=BCRC 17888T=JCM 15522T)] as the type species and Aliagarivorans taiwanensis sp. nov. [type strain is AAT1T (=BCRC 17889T=JCM 15537T)] as a second species.

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production ( p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.

Gene cloning, expression, and characterization of a beta-agarase, agaB34,from Agarivorans albus YKW-34.

A beta-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli DH5alpha as a host. The purified rAgaB34 was a beta-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at 30 degrees and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to 50 degrees . Its specific activity and kcat/Km value for agarose were 242 U/mg and 1.7x106/sM, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, beta-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34

An extracellular beta-agarase (AgaA34) was purified from a newly isolated marine bacterium, Agarivorans albus YKW-34 from the gut of a turban shell. AgaA34 was purified to homogeneity by ion exchange and gel filtration chromatographies with a recovery of 30% and a fold of ten. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40 degrees C, respectively. It was stable over pH 6.0-11.0 and at temperature up to 50 degrees C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy and (13)C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The k (cat)/K (m) values for hydrolysis agarose and neoagarotetraose were 4.04 x 10(3) and 8.1 x 10(2) s(-1) M(-1), respectively. AgaA34 was resistant to denaturing reagents (sodium dodecyl sulfate and urea). Metal ions were not required for its activity, while reducing reagents (beta-Me and dithiothreitol, DTT) increased its activity by 30%.

Agarivorans albus gen. nov., sp. nov., a gamma-proteobacterium isolated from marine animals.

Six bacterial strains were isolated from healthy marine organisms that were collected from the coast of the Kanto area in Japan. Phylogenetic analysis based on 16S rDNA sequence similarity showed that the six isolates formed a separate cluster in the gamma-Proteobacteria and were related to the genera Alteromonas and Glaciecola (<91.6 % similarity). The isolates were related closely to each other (DNA-DNA reassociation values of 74-93 %). The isolates had a polar flagellum and were Gram-negative, mesophilic, strictly aerobic rods that required salt for growth. Distinct phenotypic features of this group included the ability to hydrolyse agar and white pigmentation of colonies. The DNA G+C content of the isolates was 48-50 mol%. The major quinone was Q-8. Phenotypic characteristics of the isolates differed from those of members of the genera Alteromonas and GLACIECOLA: The name Agarivorans albus gen. nov., sp. nov. is proposed for the six isolates; the type strain is MKT 106(T) (=IAM 14998(T)=LMG 21761(T)).