Responses of <i>Pseudomonas putida</i> to Zinc Excess Determined at the Proteome Level: Pathways Dependent and Independent of ColRS

Zinc is an important micronutrient for bacteria, but its excess is toxic. Recently, the ColRS two-component system was shown to detect and respond to zinc excess and to contribute to zinc tolerance of <i>Pseudomonas putida</i>. Here, we applied a label-free whole-cell proteome analysis to compare the zinc-induced responses of <i>P. putida</i> and <i>colR</i> knockout. We identified dozens of proteins that responded to zinc in a ColR-independent manner, among others, known metal efflux systems CzcCBA1, CzcCBA2, CadA2 and CzcD. Nine proteins were affected in a ColR-dependent manner, and besides known ColR targets, four new candidates for ColR regulon were identified. Despite the relatively modest ColR-dependent changes of wild-type, <i>colR</i> deficiency resulted in drastic proteome alterations, with 122 proteins up- and 62 down-regulated by zinc. This zinc-promoted response had remarkable overlap with the alternative sigma factor AlgU-controlled regulon in <i>P. aeruginosa</i>. The most prominent hallmark was a high induction of alginate biosynthesis proteins and regulators. This response likely alleviates the zinc stress, as the AlgU-regulated alginate regulator AmrZ was shown to contribute to zinc tolerance of <i>colR</i> knockout. Thus, the ColRS system is important for zinc homeostasis, and in its absence, alternative stress response pathways are activated to support the zinc tolerance

Deep Quantitative Proteomics Reveals Extensive Metabolic Reprogramming and Cancer-Like Changes of Ectopic Endometriotic Stromal Cells

Endometriosis is a prevalent health condition in women of reproductive age characterized by ectopic growth of endometrial-like tissue in the extrauterine environment. Thorough understanding of the molecular mechanisms underlying the disease is still incomplete. We dissected eutopic and ectopic endometrial primary stromal cell proteomes to a depth of nearly 6900 proteins using quantitative mass spectrometry with a spike-in SILAC standard. Acquired data revealed metabolic reprogramming of ectopic stromal cells with extensive upregulation of glycolysis and downregulation of oxidative respiration, a widespread metabolic phenotype known as the Warburg effect and previously described in many cancers. These changes in metabolism are additionally accompanied by attenuated aerobic respiration of ectopic endometrial stromal cells as measured by live-cell oximetry and by altered mRNA levels of respective enzyme complexes. Our results additionally highlight other molecular changes of ectopic endometriotic stromal cells indicating reduced apoptotic potential, increased cellular invasiveness and adhesiveness, and altered immune function. Altogether, these comprehensive proteomics data refine the current understanding of endometriosis pathogenesis and present new avenues for therapies

An NMR and MD Modeling Insight into Nucleation of 1,2-Alkanediols: Selective Crystallization of Lipase-Catalytically Resolved Enantiomers from the Reaction Mixtures

The work on developing a scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediols, complemented by crystallization of the pure enantiomers from the reaction mixtures, offered the possibility of a more detailed study of the aggregation of such diols. MD modeling, mass spectrometry, ¹H NMR, and DOSY studies provided a novel insight into the nucleation process. An efficient protocol for stereo- and chemoselective crystallization of (<i>S</i>)-<b>1</b>,2-dodecanediol and related compounds from the crude bioconversion mixtures was developed

Individual tissue-expression of BmorCSP mRNAs.

<p>Agarose gel electrophoresis of BmorCSP1-, BmorCSP2-, BmorCSP4-, BmorCSP14-, PBP1- and Actin4-encoding cDNA PCR products from the antennae (A), legs (L), head (H), pheromone gland (P) and wings (W) from five individual newly-emerged virgin females of the silkworm moth <i>B. mori</i> (Fm1, Fm2, Fm3, Fm4 and Fm5). Genomic DNA (g) was amplified in the same conditions, showing no differences among individuals.</p

Schematic organization of BmorCSP genes.

<p>The four BmorCSPs (BmorCSP1, BmorCSP2, BmorCSP4 and BmorCSP14) matched scaffold nsaf2767 in positions 270K-360K. “BGIBMGA” are gene names identified for BmorCSPs in silkDB. Grey boxes indicate complete genes. The pointing arrow refers to the orientation of the gene (5′ to 3′). Exons are shown as black boxes and intron regions as dotted lines. Repetitive elements are shown as black-bordered white boxes. The numbers on the scale represent the position of the genes in the scaffold. <i>BmorCSP4</i> is the largest gene (1978 bps) and sits distantly from <i>BmorCSP1</i>, <i>BmorCSP2</i> and <i>BmorCSP14</i>.</p

Functional expression of CSP-RDDs.

<p>Electrophoretic separation and Western blot analysis of CSP proteins in the antennae (A), legs (L), heads (H), pheromone gland (P) and wings (W) of female <i>B. mori</i>. Protein extracts corresponding to 10 antennae, 30 legs, 5 heads, 5 pheromone glands and 20 wings equivalent were subjected to 15% SDS-PAGE (A. and D.) and transferred to nitrocellulose membranes (B–C. and E.F.). Nitrocellulose blots were labeled with two different antisera: “anti-CSP1” (B. and E.) and “antiCSP14” (C. and F.). Positions of molecular weight markers are indicated on the left of the gel. Proteins of 9 to 14 kDa are labeled with the two CSP antibodies in the pheromone gland, legs and wings samples.</p

Tissue-specific editing on cDNA of BmorCSP mRNAs.

<p>Sequence analysis of cDNA PCR products encoding BmorCSP1 reveals nucleotide insertion, deletion and substitution at the editing sites (black rectangles) in the antennae, head, legs, pheromone gland (Pgland) and wings. The size of the black rectangle is proportional to the frequency of RDDs at this location. The consensus sequence below the alignment corresponds to the nucleotide composition of the genomic DNA sequence (gDNA) encoding BmorCSP1. The number in brackets next to tissue indicates the number of CSP clones obtained for each tissue cDNA and gDNA. “.” indicates that the base is similar to the consensus sequence on this location. “A”, “T”, “G”, “C” point out a switch to adenosine, thymidine, guanosine and cytosine base in tissue-specific cDNA sequences, respectively. “1⁻” indicates base deletion in one sequence of the tissue group. “n^A” indicates switch to A in n sequences of the tissue group. Number of mismatches seen in only one tissue: 38. Number of mismatches seen in two tissues: 4.</p

Total number of RDDs on cDNA of <i>B. mori</i> CSP-RNAs depending on gene structure and tissue expression.

<p>Total number of RDDs on cDNA of <i>B. mori</i> CSP-RNAs depending on gene structure and tissue expression.</p

Amino acid mutations on BmorCSPs in the pheromone gland.

<p>Alignment shows the amino acid composition of proteins encoded by genomic DNA. Conserved amino acid residues are shown in grey. Mutations sites on the native protein are underlined. Mutant peptide motifs are shown in bold. The arrowhead indicates amino acid insertion (mainly G). The dash above residues indicates amino acid inversion. The cross (x) indicates amino acid deletion in the motif. The grey circles below the alignment indicate the position of functional elements such as α-helices <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086932#pone.0086932-Jansen2" target="_blank">[36]</a>.</p