HWSCU2301.
Host-specificity and dynamics in bacterial communities associated with bloom-forming freshwater phytoplankton
Exponentially growing phytoplankton cultures (Aulacoseira granulata, Microcystis aeruginosa, Cylindrospermopsis raciborskii) were inoculated in 4 L WC medium. For the diatom, the medium was supplemented with silica (twice the original concentration – final Na2SiO3.9 H2O concentration 56.84 mg L-1). All the procedures were done under aseptic conditions. Each culture was immediately split in 2 flasks, and either inoculated with 0.1 L (5% v/v) bacterial inoculum (non-axenic) or amended with a similar volume of the filter-sterilized control medium. Subsequently, each culture was homogenized and 150 mL aliquots were aseptically transferred to 250mL culture flasks that were incubated at 23 ± 2° C under illumination of 90±10 µmol photons m-2 s-1 in 12h:12h light-dark cycle. For each sampling day, 3 independent replicate flasks each from the bacterial treatment and the controls were analyzed. Sampling dates varied between the phytoplankton species: Aulacoseira granulata (days 2, 9 and 16 – early exponential, exponential and stationary phases), Microcystis aeruginosa (days 2, 15 and 20 - early exponential, exponential and stationary phases) and Cylindrospermopsis raciborskii (days 2, 15 and 17 - early exponential, stationary and early senescent phases). Controls were checked for axenic condition in WC p+g medium on these same days, except for C. raciborskii where only the inoculum was checked and found to be not axenic.   DNA extraction was performed as follows:  100µL of proteinase K solution (50 ng µL-1 of proteinase K in TE at pH 8) and 0.2g of glass beads (150-212 µm, Sigma-Aldrich) were added to each 1.5 mL tube.  The samples were incubated in a water bath for 15 minutes at 45°C and subsequently vortexed at 2500 rpm for 15 seconds. CTAB and NaCl was added to each tube at a final concentration of 2% CTAB and 1.35 M NaCl. The samples were incubated for 20 minutes at 65°C and 15 minutes at 55°C followed by centrifugation at 12000 x g for 15min. The supernatant was transferred to a new tube and repeatedly extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform. DNA was precipitated with cold 100% ethanol with sodium acetate (final concentration 1M) followed by a washing step with cold 70% ethanol. The DNA was resuspended to 25µL in TE-4 buffer and the DNA concentration was determined by absorbance at 260 nm on a NanoDrop 2000C spectrophotometer.  Partial bacterial 16S rRNA genes (Escherichia coli position 341-805, V3-V5 regions) was amplified using universal bacterial primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC). Primer 341F carried a 454FLX adaptor B at the 5’ end and primer 805R carried a 7-bp molecular barcode specific for each sample followed by a 454FLX adaptor A at the 5’ end.  PCR amplification was performed under the following conditions: initial denaturation at 98°C for 30s, 25 to 29 cycles at 98°C for 10s, 53°C for 30s and 72°C for 30s, and final extension at 72°C for 7min. Each sample was amplified in duplicate 30µL reactions using 10-45 ng of template DNA, 0.02 UµL-1 of Phusion DNA Polymerase (Finnzymes, Espoo, Finland), 1x Phusion HF buffer, 0.2mM dNTPs and 0.25 µM of each primer. Each amplification was checked by electrophoresis on a 1% agarose gel. The replicate PCR reactions were pooled and purified using Agencourt AMPure XP PCR purification kit according to manufacturer instructions (Beckman Coulter Inc., Brea, CA, USA). Purified amplicons were quantified using the Quant-iT PicoGreen dsDNA assay Kit (Invitrogen) and pooled in a known concentration to obtain at least 3,000 sequences for the free-living community and 9,000 sequences for attached community of each sample.  Barcodes:  HWSCU2301.sff  Bac_In,TACTCTC  A_d2_fb1,TACGTCA  A_d2_fb2,TACGAGT  M_d2_fb1,TAGACGA  M_d2_fb2,TAGAGAC  M_d2_fb3,TAGAGCA  C_d2_fb1,TAGCGTA  C_d2_fb2,TCTACTC  C_d2_fb3,TCTCTCA  A_d9_fb1,TCTGAGT  A_d9_fb2,TCATAGC  A_d9_fb3,TCATCTC  M_d15_fb1,TCACTAC  M_d15_fb2,TCACTCT  C_d17_fb1,TGTGCTA  C_d17_fb2,TGATCAC  C_d17_fb3,TGACTCA  M_d20_fb1,TGAGCAT  M_d20_fb2,TGCTAGA  M_d20_fb3,TGCTGTA  Bac_In.e1,TGCATCA  A_d10.e1_fb1,ATACGCT  A_d10.e1_fb2,ATAGCGT  A_d10.e1_fb3,ATCTCAC  A_d15.e1_fb1,ATCAGCT  A_d15.e1_fb2,ATCGTGT  A_d15.e1_fb3,ATCGACT  M_d17.e1_fb2,ATGTGCT  M_d17.e1_fb3,ATGAGTC  M_d34.e1_fb1,ACTATGC  M_d34.e1_fb2,ACTACAC  M_d34.e1_fb3,ACTACGT  M_d20_ab1,TGAGTAC  M_d20_ab2,TGAGTCT  M_d20_ab3,TGAGTGA  A_d10.e1_ab1,TGCACTA  A_d10.e1_ab2,TGCGATA  A_d10.e1_ab3,ATACTGC  A_d15.e1_ab1,ATCACTC  A_d15.e1_ab2,ATCACGT  A_d15.e1_ab3,ATCAGAC  M_d17.e1_ab2,ATCGCAT  M_d17.e1_ab3,ATGTCGT  M_d34.e1_ab1,ATGCTGT  M_d34.e1_ab2,ATGCACT  M_d34.e1_ab3,ATGCGAT  A_d15.e1_Ct,ATCATGC  M_d32.e1_Ct,ATGAGCA  C_d15_Ct,TCACACA  negative_control,ACTCTAC    HWSCU2302.sff  Bac_In,TACTCTC  A_d2_fb1,TACGTCA  A_d2_fb2,TACGAGT  M_d2_fb1,TAGACGA  M_d2_fb2,TAGAGAC  M_d2_fb3,TAGAGCA  C_d2_fb1,TAGCGTA  C_d2_fb2,TCTACTC  C_d2_fb3,TCTCTCA  A_d9_fb1,TCTGAGT  A_d9_fb2,TCATAGC  A_d9_fb3,TCATCTC  M_d15_fb1,TCACTAC  M_d15_fb2,TCACTCT  C_d17_fb1,TGTGCTA  C_d17_fb2,TGATCAC  C_d17_fb3,TGACTCA  M_d20_fb1,TGAGCAT  M_d20_fb2,TGCTAGA  M_d20_fb3,TGCTGTA  Bac_In.e1,TGCATCA  A_d10.e1_fb1,ATACGCT  A_d10.e1_fb2,ATAGCGT  A_d10.e1_fb3,ATCTCAC  A_d15.e1_fb1,ATCAGCT  A_d15.e1_fb2,ATCGTGT  A_d15.e1_fb3,ATCGACT  M_d17.e1_fb2,ATGTGCT  M_d17.e1_fb3,ATGAGTC  M_d34.e1_fb1,ACTATGC  M_d34.e1_fb2,ACTACAC  M_d34.e1_fb3,ACTACGT.  Loaded samples are      SAMN02189857: Bac_In ;     SAMN02189858: A_d2_ab1 ;     SAMN02189859: A_d2_ab2 ;     SAMN02189860: A_d2_ab3 ;     SAMN02189861: A_d2_fb1 ;     SAMN02189862: A_d2_fb2 ;     SAMN02189863: A_d9_ab1 ;     SAMN02189864: A_d9_ab2 ;     SAMN02189865: A_d9_ab3 ;     SAMN02189866: A_d9_fb1 ;     SAMN02189867: A_d9_fb2 ;     SAMN02189868: A_d9_fb3 ;     SAMN02189869: A_d16_ab1 ;     SAMN02189870: A_d16_ab2 ;     SAMN02189871: A_d16_ab3 ;     SAMN02189872: A_d16_fb1 ;     SAMN02189873: A_d16_fb2 ;     SAMN02189874: A_d16_fb3 ;     SAMN02189875: C_d2_ab2 ;     SAMN02189876: C_d2_ab3 ;     SAMN02189877: C_d2_fb1 ;     SAMN02189878: C_d2_fb2 ;     SAMN02189879: C_d2_fb3 ;     SAMN02189880: C_d15_ab1 ;     SAMN02189881: C_d15_ab2 ;     SAMN02189882: C_d15_ab3 ;     SAMN02189883: C_d15_fb1 ;     SAMN02189884: C_d15_fb2 ;     SAMN02189885: C_d15_fb3 ;     SAMN02189886: C_d17_ab1 ;     SAMN02189887: C_d17_ab2 ;     SAMN02189888: C_d17_ab3 ;     SAMN02189889: C_d17_fb1 ;     SAMN02189890: C_d17_fb2 ;     SAMN02189891: C_d17_fb3 ;     SAMN02189892: C_d2_Ct ;     SAMN02189893: C_d15_Ct ;     SAMN02189894: C_d17_Ct ;     SAMN02189895: M_d2_ab1 ;     SAMN02189896: M_d2_ab2 ;     SAMN02189897: M_d2_ab3 ;     SAMN02189898: M_d2_fb1 ;     SAMN02189899: M_d2_fb2 ;     SAMN02189900: M_d2_fb3 ;     SAMN02189901: M_d15_ab1 ;     SAMN02189902: M_d15_ab2 ;     SAMN02189903: M_d15_ab3 ;     SAMN02189904: M_d15_fb1 ;     SAMN02189905: M_d15_fb2 ;     SAMN02189906: M_d15_fb3 ;     SAMN02189907: M_d20_ab1 ;     SAMN02189908: M_d20_ab2 ;     SAMN02189909: M_d20_ab3 ;     SAMN02189910: M_d20_fb1 ;     SAMN02189911: M_d20_fb2 ;     SAMN02189912: M_d20_fb3 ;     SAMN02189913: M_d20_Ct ;     SAMN02189914: A_d16_Ct
PhytoExpInessa
Uppsala University, Department of Ecology and Gene
HWSCU2301.