Tracing evolutionary footprints to identify novel gene functional linkages.

Systematic determination of gene function is an essential step in fully understanding the precise contribution of each gene for the proper execution of molecular functions in the cell. Gene functional linkage is defined as to describe the relationship of a group of genes with similar functions. With thousands of genomes sequenced, there arises a great opportunity to utilize gene evolutionary information to identify gene functional linkages. To this end, we established a computational method (called TRACE) to trace gene footprints through a gene functional network constructed from 341 prokaryotic genomes. TRACE performance was validated and successfully tested to predict enzyme functions as well as components of pathway. A so far undescribed chromosome partitioning-like protein ro03654 of an oleaginous bacteria Rhodococcus sp. RHA1 (RHA1) was predicted and verified experimentally with its deletion mutant showing growth inhibition compared to RHA1 wild type. In addition, four proteins were predicted to act as prokaryotic SNARE-like proteins, and two of them were shown to be localized at the plasma membrane. Thus, we believe that TRACE is an effective new method to infer prokaryotic gene functional linkages by tracing evolutionary events

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans.

Lipid droplets are cellular organelles that consists of a neutral lipid core covered by a monolayer of phospholipids and many proteins. They are thought to function in the storage, transport, and metabolism of lipids, in signaling, and as a specialized microenvironment for metabolism in most types of cells from prokaryotic to eukaryotic organisms. Lipid droplets have received a lot of attention in the last 10 years as they are linked to the progression of many metabolic diseases and hold great potential for the development of neutral lipid-derived products, such as biofuels, food supplements, hormones, and medicines. Proteomic analysis of lipid droplets has yielded a comprehensive catalog of lipid droplet proteins, shedding light on the function of this organelle and providing evidence that its function is conserved from bacteria to man. This review summarizes many of the proteomic studies on lipid droplets from a wide range of organisms, providing an evolutionary perspective on this organelle

Tracing Evolutionary Footprints to Identify Novel Gene Functional Linkages

<div><p>Systematic determination of gene function is an essential step in fully understanding the precise contribution of each gene for the proper execution of molecular functions in the cell. Gene functional linkage is defined as to describe the relationship of a group of genes with similar functions. With thousands of genomes sequenced, there arises a great opportunity to utilize gene evolutionary information to identify gene functional linkages. To this end, we established a computational method (called TRACE) to trace gene footprints through a gene functional network constructed from 341 prokaryotic genomes. TRACE performance was validated and successfully tested to predict enzyme functions as well as components of pathway. A so far undescribed chromosome partitioning-like protein ro03654 of an oleaginous bacteria <i>Rhodococcus sp.</i> RHA1 (RHA1) was predicted and verified experimentally with its deletion mutant showing growth inhibition compared to RHA1 wild type. In addition, four proteins were predicted to act as prokaryotic SNARE-like proteins, and two of them were shown to be localized at the plasma membrane. Thus, we believe that TRACE is an effective new method to infer prokaryotic gene functional linkages by tracing evolutionary events.</p></div

Plasma membrane location of SNARE-like proteins ro03137 and ro05535 in RHA1 cells.

<p>(A) Predicted domains of gene 111022500, 111022501, 111020126 and 111025334. Gene 111022500 and 111022501 were fused into one operon construct. They both include SNARE_assoc domains (PF09335) predicted by the PFAM database. (B) The minimum evolution tree shown for genes 111022500, 111022501, 111020126 and 111025334. The evolution distance (in billion years) and tree was calculated and constructed by using the maximum parsimony (MP) method embedded in MEGA4.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066817#pone.0066817-Tamura1" target="_blank">[54]</a>. (C) SNARE-like protein-coding genes ro03137 and ro05535 were amplified and inserted into the over-expression vector pJAM2<i>-egfp</i> to generate pJAM2-<i>ro03137-egfp</i> and pJAM2-<i>ro05535-egfp</i>. The plasmids were transformed into RHA1 WT cells by electroporation. Single colonies were picked, cultivated in LB for 48 h and transferred for cultivation into MSM for 24 h. Lipid droplets were stained by LipidTOX as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066817#pone.0066817-Ding2" target="_blank">[36]</a>. Cells were prepared for confocal microscopy as described in Materials and Methods. (a1–a4), over-expression of empty vector pJAM2-<i>egfp</i>; (b1–b4), over-expression of pJAM2-<i>ro03137-egfp</i>; (c1–c4), over-expression of pJAM2-<i>ro05535-egfp</i> (Bar  = 10 µm).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Growth rate comparison of RHA1 WT and a <i>r-parB</i> deletion mutant strain.

<p>(A) Growth curves for wild type (red curve) and <i>r-parB</i> deletion mutant (blue curve) in LB medium. (B) Protein content for RHA1-WT and <i>r-parB</i> deletion mutant. Bacterial samples were collected at different time points and washed twice with PBS before being dissolved in 1% Triton X-100 followed by sonication. Quantification of protein and TAG content was performed as described in Material and Methods. (C) TAG content of RHA1-WT and <i>r-parB</i> deletion mutant strains; identical amounts of cells were transferred into MSM medium after pre-culturing in LB. (D) TAG/protein content ratio for the two strains. (E) TAG was extracted from the same cell number, with cells cultivated in MSM for 24 h prior to TLC analysis. M, marker; lane 1, <i>r-parB</i> deletion mutant; lane 2, <i>r-parB</i> deletion mutant with pJAM2<i>-r-parB-egfp</i> over-expression plasmid; lane 3, RHA1-WT; lane 4, RHA1-WT with pJAM2<i>-parB-egfp</i> over-expression plasmid.</p

Schematic view of the TRACE method.

<p>Prokaryotic genomes were selected from each genus in order to ensure evolutionary distances. A gene functional network was constructed by defining the similarity weight and operon weight. The shortest path values were then calculated from seed genes to each candidate gene. (i) shows functional connections passed on by three genes o-1, o-2, o-3 in an operon. (ii) shows functional connections passed on by two domains, d-1 and d-2. Whole genome genes were ranked by shortest path distances and the highest-ranked gene was considered to be its functional linkage gene.</p

Predictive power of TRACE for enzyme function and components of pathways.

<p>(A) Validation of predicting enzyme function. (B) Validation results of predicting components of pathway. For both figures, ROC curves of TRACE were obtained for both random genes set and whole genome set. The results were compared between TRACE and PPM. In addition, the predictive power of TRACE was validated on a permutated network using both control sets.</p

Computational analysis and domain prediction for R-ParB protein.

<p>(A) The top three ranked genes of <i>r-parB</i> and their connection details. In the connected paths, two operon genes 50955949 and 50955950 served as a key bridge link (red arrow). The identified three genes (111020644, 111019886, 111017945) exhibit sequence similarity between each other (green dotted line), but not with <i>r-parB</i>. The probability between genes in the same operon and sequence similarity (e-values retrieved from BLASTP) are presented. The genes and related genomes in connected paths are depicted in identical colours. (B) The predicted protein domains for gene 111020643 using the NCBI CDD and PFAM databases. There are two overlapping domains (blue) predicted by NCBI CDD database and two spatial separated domains (red) by PFAM database. The e-values were calculated by both databases.</p

Morphology of the <i>parB</i> deletion mutant strain.

<p>(A) Transmission electron microscopy on RHA1 WT cells and <i>r-parB</i> deletion mutant strain. a, negative staining of RHA1-WT; b, negative staining of <i>r-parB</i> deletion mutants; c and d were the enlarged images of a and b, respectively. Bar  = 2 µm. (B) Identification of <i>r-parB</i> deletion mutant. a, PCR result of <i>r-parB</i> gene deletion. M, marker; lane 1, positive control of <i>r-parB</i> gene PCR fragment using primers r-parB-a/r-parB-d with knockout plasmid pK18mobsacB as template; lane 2, the PCR fragment of <i>r-parB</i> gene in the deletion mutant was 848 bp, and identical to the positive control; lane 3, the size of the WT fragment was 1930 bp; lane 4–5, the left flank sequences of AB were 410 bp used primers r-parB-a/r-parB-b with WT and deletion mutant cells as templates, respectively; lane 6–7, the right flank sequences of primers r-parB-c/r-parB-d, with templates as in lane 4–5; lane 8–9, r-parB-f and r-parB-r primers in <i>r-parB</i> gene sequence. b, diagram of <i>r-parB</i> gene deletion. Primers used as shown.</p