Biocontrol Science, 2020, Vol. 25, No. 1, 25—33 Original Diversity of the Photosynthetic Bacterial Communities in Highly Eutrophicated Yamagawa Bay Sediments ISLAM TEIBA1,3, TAKESHI YOSHIKAWA2＊, SUGURU OKUNISHI2, MAKOTO IKENAGA4, MOHAMMED EL BASUINI2,3, HIROTO MAEDA2 1 The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-8580, Japan 2 Research Field in Fisheries; Agriculture, Fisheries and Veterinary Medicine Area; Research and Education Assembly; Kagoshima University; 4-50-20 Shimoarata, Kagoshima 890-0056, Japan 3 Faculty of Agriculture, Tanta University, Sebrbay, Tanta, El-Gharbia Governorate, Arab Republic of Egypt 4 Research Field in Agriculture; Agriculture, Fisheries and Veterinary Medicine Area; Research and Education Assembly; Kagoshima University; 1-21-24 Korimoto, Kagoshima 890-8580, Japan Received 24 September, 2019/Accepted 5 November, 2019 Yamagawa Bay, located in Ibusuki, Kagoshima Prefecture, Japan, is a geographically enclosed coastal marine inlet, and its deteriorating seabed sediments are under an anoxic, reductive, sulfide-rich condition. In order to gain insight into diversity of anoxygenic photosynthetic bacteria（ AnPBs） and their ecophysiological roles in the sediments, three approaches were adopted: isolation of AnPBs, PCR-DGGE of 16S rDNA, and PCR-DGGE of pufM. Among the bacterial isolates, relatives of Rhodobacter sphaeroides were most dominant, possibly contributing to transforming organic pollutants in the sediments. Abundance of Chlorobium phaeobacteroides BS1 was suggested by 16S rDNA PCR-DGGE. It could reflect intensive stratification and resultant formation of the anoxic, sulfide-rich layer in addition to extreme low-light adaptation of this strain. Diverse purple non-sulfur or sulfur bacteria as well as aerobic anoxygenic photoheterotrophs were also detected by pufM PCR-DGGE, which could be associated with organic or inorganic sulfur cycling. The outcome of the present study highlights ecophysiologically important roles of AnPBs in the organically polluted marine sediments. Key words : 16S rDNA / Anoxygenic photosynthetic bacteria / Eutrophicated marine sediment / PCR- DGGE / pufM. INTRODUCTION bottom-water hypoxia and sulfide accumulation in the sediment（ Ide, 2012）. Coastal aquatic systems are highly susceptible and Aquatic microbiota vary spatio-temporarily due to can be directly or indirectly affected by adjacent terres- changes of their surrounding environment, and compo- trial ecosystems, anthropogenic activities and climate sition and dominancy of the bacterial assemblages are change. Yamagawa Bay is a coastal basin located in highly correlated to the environmental conditions; thus, Ibusuki, Kagoshima Prefecture, Japan with a central elucidation of the community structure enable us to depth of up to 50 m with a shallow entrance of 8 m. The understand physicochemical status of the environments, geographical feature gives rise to reduction of the water especially of the sediments. exchange and makes the water stagnant for a long time, Anoxygenic photosynthetic bacteria（ AnPBs） are allowing deposition of organic matters and resulting in Gram-negative prokaryotes, performing anoxygenic photosynthesis with pigments such as bacteriochloro- ＊Corresponding author. Tel: +81-99-286-4191, Fax: +81-99- phylls（ Bchl） and carotenoids. They convert light energy 286-4015, E-mail : yoshi（a）fish.kagoshima-u.ac.jp into chemical energy and grow autotrophically by using 26 T. YOSHIKAWA ET AL. carbon dioxide as a sole source of carbon. Major groups 0.1; Na2MoO4·2H2O, 0.02; shown as grams per 100 are purple non-sulfur bacteria, purple sulfur bacteria, mL）, 1mL/L. green sulfur bacteria and green non-sulfur bacteria For AnPB isolation, a double layer agar technique （Koblížek et al., 2006）. Aerobic anoxygenic phototro- was used by spreading the enriched cultures on Basic phic bacteria, accounting for up to 10% of bacterial I plates with 1.5% of agar and then covering the plates communities in the marine euphotic zones（ Yutin et with 1.2% agar. The agar plates were incubated anaer- al., 2007）, also produce Bchl a and complement their obically with Anaeropack Kenki system（ Mitsubishi Gas energy requirements by harvesting light under an aerobic Chemical, Tokyo, Japan） under the same condition condition. Habitats of AnPBs are restricted by availabil- as above. Pure isolates were obtained by sequential ity of light and electron donors including reduced sulfur isolation from colonies with different morphology and or organic compounds for their phototrophic growth maintained in Basic I liquid or agar plate media for further as well as redox potential（ van Gemerden and Mas, application. 1995; Guyoneaud et al., 1996）. Therefore, community structure of the phototrophs will be a good bioindica- Determination of the 16S rDNA nucleotide sequences tor reflecting their ambient pollution levels, especially Liquid cultures of the isolates were centrifuged to in the organically polluted marine sediments, resulting obtain cell pellets, from which the bacterial DNA were in eutrophication, oxygen depletion and high sulfide extracted with DNeasy Plant Mini Kit（ Qiagen, Hilden, concentration. Germany）. Genes of 16S rRNA were amplified by PCR The aim of this study is to gain insight into diver- using a universal primer set 27F and 1525R（ TABLE sity of AnPBs in the Yamagawa Bay sediments. Three 1）. A reaction mixture of PCR consisted of 1 x ExTaq approaches were adopted: isolation and identification Buffer（ Takara Bio, Otsu, Japan）, 100 µM dNTP Mixture of pigmented anaerobic microorganisms; polymerase （Takara Bio, Otsu, Japan）, 0.5 µM primers and 0.025 chain reaction-denaturing gradient gel electrophore- units·µL-1 ExTaq DNA Polymerase（ Hot Start Version, sis（ PCR-DGGE） of 16S ribosomal RNA（ 16S rRNA） Takara Bio, Otsu, Japan）, and 5 µL of the bacterial DNA genes（ 16S rDNA）; and PCR-DGGE of pufM encoding solutions were added to 100 µL of the mixture. Thermal the M subunit of the reaction center complex. cycling was conducted at 94˚C for 1 min, followed by 25 cycles of denaturation at 94˚C for 30 s, annealing at MATERIALS AND METHODS 58˚C for 30 s and extension at 72˚C for 90 s, and final extension at 72˚C was performed for 7 min. Specific Sample collection amplification of the target gene was confirmed by Sediment samples were collected from Yamagawa subjecting the PCR products to 1.5% agarose gel elec- Bay, Kagoshima, Japan from May to November 2016 trophoresis in TAE buffer（ 40 mM Tris-acetate, pH 8.3, 1 and May 2017 with a G.S. type core sampler（ Ashura）. mM ethylenediaminetetraacetic acid）. Surface sediments within a depth of 10 mm were The amplified 16S rDNA fragments were cleaned up used for bacterial isolation and environmental DNA with ExoSAP-IT（ Affymetrix, Santa Clara, CA, USA）, and preparation. their nucleotide sequences were determined using a set of universal primers, 27F, PrSSU.2F, 1525R and 531R Enrichment and isolation of photosynthetic bacteria （TABLE 1）, with ABI PRISM BigDye Terminator v3.1 Portions of the collected sediments were transferred Cycle Sequencing Kit（ Applied Biosystems, Waltham, into tightly-sealed test tubes filled with 30 mL of Basic MA, USA）. The obtained sequences were assembled I medium（ Hoshino and Kitamura, 1984） and culti- with the program GENETYX-MAC Ver. 19（ Genetyx, vated at 20˚C under 12:12 light:dark cycling condition Tokyo, Japan）. Their most homologous sequences were in order to enrich photosynthetic bacteria. Composition retrieved from the GenBank DNA database with the of the medium is as follows（ concentrations are given program of Basic Local Alignment Search Tool（ BLAST; as grams per liter except as otherwise noted）: KH2PO4, Altschul et al., 1990）. 0.5; K2HPO4, 0.6;（ NH4）2SO4, 1.0; MgSO4·7H2O, 0.2; NaCl, 0.2; CaCl2·2H2O, 0.05; Na2S2O3·5H2O, 0.1; yeast Bacterial community analyses by 16S rDNA PCR- extract, 0.1; malate, 0.5. The following supplements DGGE were also added: the growth factor solution（ thiamin-HCl, Microbial DNAs in the sediments were extracted using 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.03; PowerSoil DNA Isolation Kit（ MOBio, Carlsbad, CA, vitamin B12, 0.01; pyridoxine-HCl, 0.01; D-biotin, 0.005; USA）. Amplification of their 16S rDNAs was conducted shown as grams per 100 mL）, 1 mL/L; the trace by PCR with the primers 341F-GC and 907R（ TABLE element solution（ EDTA·2Na, 2.0; FeSO4·7H2O, 2.0; 1）; composition of the reaction mixtures was the same H3BO3, 0.1; CoCl2·6H2O, 0.1; ZnCl2, 0.1; MnCl2·4H2O, as above. After initial denaturation at 95˚C for 1 min, PHOTOTROPHS IN MARINE SEDIMENT （ for theprimersetwithaGCclamp:annealingtempera The samethermalsettingasabovewasadopted,except consortia with the color of green, pink or yellow. Totally Totally pinkoryellow. consortia withthecolorof green, ofmicrobial conditionshowedgrowth or anaerobic ments intheBasicIliquidmedium undersemi-anaerobic Isolation andidentification ofphotosyntheticbacteria performed. were and theirhomologysearches determinedusing theprimerpufM557F were sequences Nucleotide analyses. further for excluded were stillheterogeneous bands whosesequenceswere obtained; untilhomogeneous sequenceswere repeated was ers, andsubjectedtoDGGE.Thisprocedure same prim with the excised, re-amplified were bands 30-60% or40-60%ofthedenaturant.Representative polyacrylamide and 10% of shown above, exceptfor respectively. 58-63˚Cand10-12cycles, andcyclenumberswere ture pufM750R, orpufM557FGCandpufM750R the secondrunwithinnerprimersetspufM557Fand furthersubjectedto