Characterization and Modeling of Thermostable GH50 Agarases from Microbulbifer elongatus PORT2

Viewing the considerable potential of marine agar as a source for the sustainable production of energy as well as nature-derived pharmaceutics, this work investigated the catalytic activity of three novel GH50 agarases from the mesophilic marine bacterium Microbulbifer elongatus PORT2 isolated from Indonesian coastal seawaters. The GH50 agarases AgaA50, AgaB50, and AgaC50 were identified through genome analysis; the corresponding genes were cloned and expressed in Escherichia coli BL21 (DE3). All recombinant agarases hydrolyzed β-p-nitrophenyl galactopyranoside, indicating β-glycosidase characteristics. AgaA50 and AgaB50 were able to cleave diverse natural agar species derived from Indonesian agarophytes, indicating a promising tolerance of these enzymes for substrate modifications. All three GH50 agarases degraded agarose, albeit with remarkable diversity in their catalytic activity and mode of action. AgaA50 and AgaC50 exerted exolytic activity releasing differently sized neoagarobioses, while AgaB50 showed additional endolytic activity in dependence on the substrate size. Surprisingly, AgaA50 and AgaB50 revealed considerable thermostability, retaining over 75% activity after 1-h incubation at 50 °C. Considering the thermal properties of agar, this makes these enzymes promising candidates for industrial processing.

Marine bacterium Seonamhaeicola algicola strain CC1 as a potential source for the antioxidant carotenoid, zeaxanthin

Currently, there are only six species in the genus Seonamhaeicola, i.e., Seonamhaeicola aphaedonensis, S. algicola, S. marinus, S. acroporae, S. maritimus, and S. sediminis. These bacteria have typical yellow or orange color. Among the identified strains, only S. marinus that had been reported to have a yellow polyene flexirubin pigment. However, the presence of carotenoid pigments has not been reported in this genus. Recently, we successfully isolated a new strain, S. algicola strain CC1, bacterium that was found in association with a red seaweed, Halymenia sp., collected from the coast of South Malang, Indonesia. The strain was grown well in the Zobell marine agar 2216E producing yellowish pigments. According to the 16S rRNA sequencing analysis and BLAST search, the strain is closely related to S. algicola strain Gy8, with 99.78% identity. The pigment composition was separated and analyzed by a high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and the strain was found to produce zeaxanthin as the major component, which appeared at a retention time (tR) of 28.89 min, showing a typical mass spectrum with a molecular ion at m/z 568.5 [M]+ and four product ions at m/z 261.4 [M−307]+, 476.6 [M−92]+, 429.3 [M−139]+, and 536.5 [M− 32]+. Other carotenoids, including zeaxanthin cis isomers, β-cryptoxanthin, β-carotene cis isomer, and β-carotene, are as minor components. The novel and noteworthy finding of this report is the identification of a Seonamhaeicola species that produces carotenoids and can be used as a source of zeaxanthin.

Combining Culture-Dependent and Culture-Independent Methods: New Methodology Insight on the Vibrio Community of Ruditapes philippinarum

Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio species is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (culture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was investigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabarcoding detected the cultivable taxa, including rare species. Moreover, marine agar medium was found to be a useful substrate for the recovery of several Vibrio species, including the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.

Isolation, Selection and Identification of Polyaromatic Hydrocarbons (PAHs) Degrading Bacteria from Heavy Oil Waste (HOW)-Contaminated Soil

The heavy oil waste (HOW) containing polyaromatic hydrocarbon (PAHs) is a persistent organic pollutants (POPs) that difficult to degrade. The new PAH degrading consortium was investigated from HOW contaminated soil in North Sumatera of Indonesia. The isolation, selection and identification of polyaromatic hydrocarbon degrading bacteria from soil contaminated by HOW was conducted to solve a bioremediation process. The isolation microbes from soil contaminated by HOW was performed using a minimum ONR7a media and followed on marine agar media for purification purposes. From the performed isolation results, 11 isolates were able to degrade PAHs compounds, such as phenanthrene, dibenzothiophene, or fluorene compounds. They grew at pH range of 4.8-8.2 and performed on emulsification activity in paraffin from 0.150-0.662. Three of them showed the best performance on HOW biodegradation capability and then successfully selected and identified as Salipiger sp., Bacillus altitudinis, and Ochrobactrum anthropi. using 16S rDNA. The HOW biodegradation as TPH-degradation were 38.66%, 59.60%, and 47.16%, respectively. Those isolated bacteria could potentially be as bioremediation agents to develop on bioremediation process for soils contaminated by HOW.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.

Xanthomarina gelatinilytica gen. nov., sp. nov., isolated from seawater

A novel Gram-stain-negative, rod-shaped, yellow-pigmented, non-sporulating, non-motile bacterium, designated strain AK20T, was isolated from seawater collected from Kochi city, Kerala state, India. Colonies on marine agar were circular, yellow, shiny, translucent, 2–3 mm in diameter, convex and with entire margin. Flexirubin-type pigment was present. The fatty acids were dominated by iso-branched units with a high abundance of iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and iso-C15 : 0 3-OH. Polar lipids included phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and four unidentified lipids. Menaquinone 6 (MK-6) was the predominant respiratory quinone. The DNA G+C content of strain AK20T was 38.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AK20T was closely related to Formosa spongicola A2T and Bizionia paragorgiae KMM 6029T (pair-wise sequence similarities of 95.9 and 95.7 %, respectively), forming a distinct branch within the family Flavobacteriaceae and clustering with the clade comprising species of the genus Bizionia. Based on phenotypic and chemotaxonomic characteristics and phylogenetic analysis, strain AK20T is different from the existing genera in the family Flavobacteriaceae, and is therefore considered to represent a novel species of a new genus, for which the name Xanthomarina gelatinilytica gen. nov., sp. nov. is proposed. The type strain of Xanthomarina gelatinilytica is AK20T ( = MTCC 11705T = JCM 18821T).

Bizionia fulviae sp. nov., isolated from the gut of an egg cockle, Fulvia mutica

A novel Gram-staining-negative, non-spore-forming, non-flagellated, non-motile, aerobic, saffron-coloured, rod-shaped bacterium that did not produce flexirubin-type pigments was designated strain EM7T and was distinct from other members of the genus Bizionia by produce carotenoid-type pigments and being able to grow independently of NaCl. Strain EM7T was isolated from the intestinal tract of an egg cockle, Fulvia mutica, which had been collected from the West Sea in Korea. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain EM7T belonged to the genus Bizionia, and showed sequence similarity to Bizionia paragorgiae KMM 6029T (97.9 %) and Bizionia saleffrena HFDT (97.73 %). Growth occurred on marine agar 2216 at 0–25 °C (optimum, 20 °C) and at pH 6–9 (optimum, pH 7). Growth occurred in the presence of 0–10 % (w/v) NaCl (optimum, 2 %, w/v, NaCl). The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C15 : 1 G, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and iso-C16 : 0 3-OH. The major respiratory quinone was menaquinone MK-6. The polar lipids of strain EM7T comprised phosphatidylethanolamine, three unidentified aminolipids, an unidentified aminophospholipid and two unidentified lipids. The genomic DNA G+C content was 34.8 mol%. Bizionia paragorgiae KMM 6029T and Bizionia saleffrena HFDT to Bizionia paragorgiae KCTC 12304T and Bizionia saleffrena CIP 108534T, respectively. Thus, it is proposed that the isolate represents a novel species, Bizionia fulviae sp. nov., with strain EM7T ( = KACC 18255T = JCM 30417T) as the type strain.

ISOLASI DAN KARAKTERISASI BAKTERI PENGHASIL SENYAWA ANTIBAKTERI YANG BERASOSIASI DENGAN KARANG BATU DARI PERAIRAN BITUNG DAN SPONS DARI SELAT MAKASSAR

ABSTRACT Recently the needs of antibacterial compounds is increasing. This is due to the bacterial resistence to common antibacterial compounds. coral and sponge-ssociated bacteria are potential producer of antibacterial compounds. This research was aim to obtain coral and sponge-associated bacteria that could produce antibacterial compound. coral associated-bacteria was isolated from Bitung and was isolated in Marine Agar by pour plate method. The antibacterial compounds were obtained by extraction using ethyl acetate and acetone. The antibacterial assay was performed by agar diffusion method using paper discs and was performed by testing with Staphylococcus aureus, Bacillus subtilis, Vibrio cholerae biotipe El Tor, and Escherichia coli. Total 37 isolate was isolated from corals and 25 isolate from sponge obtained from Selat Makassar. Based on the assay, only bacteria from sponge that showed antibacterial activity. Two sponge-associated bacteria, S.5-8 and S.2-1 NRBC were found to inhibit S. aureus. From those isolates, isolate S.5-8 produced bigger clear zone (2,6 mm) than S.2-1 NRBC (1,5mm). S.5-8 could hydrolize gelatine whereas S.2-1 NRBC showed positive result on oxidase test and was able to fement xilose and arabinose to produce acid. Key words: antibacterial activity, association, characterization, coral, isolation, sponge

Algoriphagus faecimaris sp. nov., isolated from coastal sediment

A Gram-negative, non-motile, non-sporulating bacterial strain, designated LYX05T, was isolated from coastal sediment of Qingdao, China, on the coast of the Yellow Sea. Strain LYX05T was aerobic and heterotrophic. The strain grew optimally at 37 °C and pH 7.5 and in the presence of 2 % (w/v) NaCl. Colonies were 1–2 mm in diameter, circular, reddish orange and shiny with entire edges on marine agar medium. Cells were rods (0.3–0.5 µm wide and 0.8–1.6 µm long). The dominant fatty acids were iso-C15 : 0 (40.82 %) and C16 : 0 (10.45 %). The DNA G+C content was 42.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LYX05T was phylogenetically related to the members of the genus Algoriphagus and the closest relative was Algoriphagus hitonicola 7-UAHT (95.8 % 16S rRNA gene sequence similarity). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain LYX05T was considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus faecimaris sp. nov. is proposed. The type strain is LYX05T ( = JCM 16561T = DSM 23095T = LMG 25474T).