The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta(Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Lertransgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.

Peptide natural products exhibit broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce Natural Product Peptidogenomics (NPP), a new mass spectrometry-guided genome mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo MSn structures to genomics-based structures following current biosynthetic logic. In this study we demonstrate that NPP enabled the rapid characterization of >10 chemically diverse ribosomal and nonribosomal peptide natural products of novel composition from streptomycete bacteria as a proof of concept to begin automating the genome mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which from well-characterized model streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.

Metabolic exchange between an organism and the environment, including interactions with neighboring organisms, is important for processes of organismal development. Here we develop and use thin-layer agar natural product MALDI-TOF imaging mass spectrometry of intact bacterial colonies grown on top of the MALDI target plate to study an interaction between two species of bacteria and provide direct evidence that a Bacillus subtilis silences the defensive arsenal of Streptomyces coelicolor.

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC 50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.etabolic exchange describes the process of exchanging signals or nutrients between cells or populations and is a common feature of all living systems. Bacteria produce a wide array of signaling molecules to control metabolic as well as morphological and developmental changes in either an interspecies or intraspecies manner (1). Bacillus subtilis, for example, has a complex life cycle and thrives in diverse living conditions ranging from soil, contaminated wounds, and the intestinal tract (2-4). To accommodate this, B. subtilis dedicates ∼10% of its genome to the production of specific molecules involved in intra-and interspecies metabolic exchange (5). Two of these molecules are sporulation delaying protein (SDP) and sporulation killing factor (SKF), which, based on genetic experiments, are proposed to lyse a subpopulation of B. subtilis cells to provide nutrients for the remaining cells, a process referred to as bacterial cannibalism (6-10). This behavior is dependent on Spo0A, a master transcriptional regulator that also controls biofilm formation and sporulation (6-13).We set out to characterize these cannibalistic compounds to establish their roles in the B. subtilis life cycle and to understand their structure ...