strain PD630 (PD630), is an oleaginous bacterium, and also is one

strain PD630 (PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development. INTRODUCTION Lipid droplets (LDs) are cellular organelles widely found in fungal, plant, animal and human cells (1C3). They are encapsulated by a phospholipid monolayer and are compositionally different from other membrane structures (4). They differ in that their primary role is lipid storage, but may also be pivotal in cellular communication with organelles such as the mitochondria to regulate energy metabolism and substrate utilization. LD is an important organelle related to human metabolic diseases and biofuel productions. For example, LD dysfunction is one of the main causes of metabolic disorders such as obesity, insulin resistance, type 2 diabetes, and cardiovascular diseases (5C9). In biofuel CGS 21680 HCl studies, triacylglycerol (TAG) in LD of green algae has been investigated and developed for high oil yields by using targeted metabolic engineering (10C12), making it a biological candidate for biofuel production. Delineating the molecular mechanisms of LD dynamics is essential to understand its formation, functions, synthetic engineering and further biofuel applications. Since PD630 has the ability to accumulate large amounts of TAG in the LD (25). The importance of strain PD630 (PD630) as a model system is also exemplified by its powerful ability to convert carbon sources into lipids. Interestingly, the TAG storage in PD630 accounts for up to 87% of the cellular dry weight (26), and thus has higher lipid storage capacity when compared with other oleaginous organisms (26,27). Early studies reported that PD630 has 10 diacylglycerol acyltransferases (DGAT) that assimilate cellular fatty acids into TAG (13,28). Holder PD630. Therefore, to facilitate the application of PD630 LD production for biofuel development, a complete genome of the organism and integrated analysis of its transcriptome, a proteome of its lipid synthesis, storage and metabolism are essential. We performed multi-omic studies and present herein the complete genome sequence, a comparative transcriptome and a comparative LD proteome of PD630. After integrating the collected data, a number of protein families involved in LD dynamics were identified including lipid synthesis, LD structure-like proteins, dynamin-like and SNARE-like proteins. A structure-like protein LPD06283 was verified by its LD location and its effect CGS 21680 HCl on LD size. Together, these omics are useful tools to investigate the mechanisms of LD dynamics that will enhance our understanding of the lipid storage of LD in biofuel development. CGS 21680 HCl MATERIALS AND METHODS DNA extraction and genome sequencing and assembly Cells of PD630 (30) were obtained from Dr Steinbchels lab at the University of Mnster. Cells were cultured aerobically in 100 ml of nutrient broth (NB) at Rabbit Polyclonal to Met (phospho-Tyr1234) 30C to CGS 21680 HCl postlogarithmic phase, and then the DNA was extracted. The complete nucleotide sequence was obtained using a combination of paired-end/mate-pair Illumina sequencing, and 454 sequencing. The sequence gaps were completed by direct sequencing of polymerase chain reaction (PCR)-amplified fragments. For 454 pyrosequencing, genomic DNA was sheared up by nebulization into random fragments of 500C800 bp for the construction of a dispersed library, which was then clonally amplified and sequenced on a 454 Genome Sequencer. For Illumina sequencing, genomic DNA was processed to construct paired-end libraries with size spans of 300 bp, and also mate-pair libraries with size spans of CGS 21680 HCl 3 kb using an Illumina Genomic DNA Sample Prep kit. The total number of 454 reads obtained was 861 751, giving a 36-fold coverage, while the total number of paired-end and mate-pair library reads was 40 110 584, giving a 445-fold coverage. We used two assembly programs and combined the primary contigs and paired-end data to build scaffolds in successive assemblies. Four hundred fifty-four sequences were assembled using the Roche GS assembler, Newbler (version 2.5), with default parameters. The primary contigs were then scaffolded with Illumina mate-pair reads using SSPACE-premium (version 2.1) (31). To close the gaps among scaffolds, read pairs that were uniquely mapped to the contig tails were extracted for manual assembly. Primers were.