Biosynthesis pathways of 2-MIB in actinomycetes.

<p>Geranyl pyrophosohate (GPP), the universal precursor of monoterpenes, is converted to 2-MIB through two steps: methylation of GPP and cyclization of methyl-GPP subsequently.</p

Transcriptional changes in 2-MIB genes under darkness.

<p>The levels of <i>mtf</i> and <i>mic</i> expression under 72 h darkness were calculated relative to the expression in control culture (light density as 30 µmol photons·m⁻²·s⁻¹).</p

GC-MS analysis of volatile compounds obtained from the gaseous phase of the <i>Pseudanabaena</i> sp. dqh15 culture.

<p>Mass spectrum revealed that the main volatile compound (retenion time as 11.64 min, m/z 168) is 2-MIB.</p

2-MIB biosynthesis associated genes identified from cyanobacterial strains.

<p>A: <i>Pseudanabaena</i> sp. dqh15; B: <i>Planktothricoides raciborskii</i> CHAB 3331. The <i>cnb</i>, <i>mtf</i> and <i>mic</i> reprents cyclic nucleotide-binding protein gene, methyltransferase gene and 2-MIB cyclase gene respectively. These genes form putative operon in chromosome.</p

Organization of genes associated with 2-MIB biosynthesis in different organisms.

<p>The grayed, oblique-line, filled and opened arrows indicate <i>cnb</i>, <i>mtf</i>, <i>mic</i> and other predicted functional genes respectively.</p

Expression changes of 2-MIB associated genes under different light intensities.

<p>The levels of <i>mtf</i> and <i>mic</i> expression in different light density (10 and 60 µmol photons·m⁻²·s⁻¹) were calculated relative to the expression in control culture (30 µmol photons·m⁻²·s⁻¹).</p

Alignment of amino acid sequences of 2-MIB cyclases.

<p>Mic-PS and Mic-PL representing proteins from <i>Pseudanabaena</i> sp. dqh15 and <i>Planktothricoides raciborskii</i> CHAB 3331 respectively; Sco7700, Sgr1269, Scab5041, Ndas2620, Caci4612, and Snas1991 represent proteins from <i>Streptomyces coelicolor</i> A3(2) (AL93912), <i>S. griseus</i> NBRC 13350 (AP009493), <i>S. scabiei</i> 87.22 (FN554889), <i>Nocardiopsis dassonvillei</i> DSM 43111 (CP002040), <i>Catenulispora acidiphila</i> DSM 44928 (CP001700), and <i>Stackebrandtia nassauensis</i> DSM 44728 (CP001778), respectively. Black boxed residues show the possible Mg²⁺ binding sites; sites marked by * and # at the bottom indicate putative substrate binding pockets and active site lid residues respectively.</p

Unrooted neighbor-joining (NJ) phylogenetic trees of 2-MIB associated genes.

<p>A: The phylogenetic tree based on putative <i>mic</i> genes; B: The tree based on <i>mtf</i> genes; C: The tree based on <i>cnb</i> genes. Bootstrap values (>50%) are displayed at the nodes. Accession numbers: <i>Am. mediterranei</i> U32 (CP002000); <i>Ca. acidiphila</i> DSM 44928 (CP001700); <i>No. dassonvillei</i> DSM 43111 (CP002040); <i>P. fluorescens</i> Pf0-1 (CP000094); <i>Sa. erythraea</i> NRRL2338 (AM420293); <i>St. nassauensis</i> DSM 44728 (CP001778); <i>S. ambofaciens</i> ATCC 23877 (AM238663); <i>S. bingchenggensis</i> BCW-1 (CP002047); <i>S. coelicolor</i> A3(2) (AL939132); <i>S. griseus</i> NBRC 13350 (AP009493); <i>S. lasaliensis</i> ATCC 31180 (AB547324); <i>S. scabiei</i> 87.22 (FN554889); <i>Cyanothece</i> sp. PCC 7424 (CP001291); <i>Cyanothece</i> sp. PCC 7425 (CP001344).</p

Table_1_Characterization of Microcystis (Cyanobacteria) Genotypes Based on the Internal Transcribed Spacer Region of rRNA by Next-Generation Sequencing.DOCX

<p>Microcystis is one of the most common and dominant bloom-forming cyanobacteria in freshwater worldwide. The method for genotype detection based on traditional molecular cloning is expensive and time consuming and generates a limited number of sequences. In this study, a high-throughput sequencing (HTS) method was developed to detect the internal transcribed spacer (ITS) regions between 16S and 23S rRNA region of Microcystis populations along a typical water system in Yuqiao Reservoir–Haihe River in Tianjin, northern China. A total of 629,341 reads were obtained and clustered into 2005 operational taxonomic units (OTUs). Analysis of alpha diversity indices showed that the Haihe River is more diverse than Yuqiao Reservoir. In general, the two water areas exhibit a clear differentiation pattern in OTU abundance, sharing genotypes from a small part of Yuqiao Reservoir with those in the Haihe River. Phylogenetic analysis further indicated the possible flexible evolution of Microcystis genotypes occurring in the research areas. This study provides the first exhaustive description of HTS method for detection of ITS region to evaluate Microcystis intra-species diversity and relationship.</p