An anthocyanin marker for direct visualization of plant transformation and its use to study nitrogen-fixing nodule development.

The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis

Complete Genome Sequence of Propionibacterium avidum Strain 44067, Isolated from a Human Skin Abscess

Propionibacterium avidum is an anaerobic Gram-positive bacterium that forms part of the normal human cutaneous microbiota, colonizing moist areas such as the vestibule of the nose, axilla, and perineum. Here we present the complete genome sequence of P. avidum strain 44067, which was isolated from a carbuncle of the trunk

An acidophilic bacterial-archaeal-fungal ecosystem linked to formation of ferruginous crusts and stalactites.

The presence of specialized microbial associations between populations of chemoautotrophic bacteria and archaea with ascomycetous fungi was observed inside stalactite-shaped mineral formations in a highly acidic cave environment. Metagenomic, chemical and electron microscopy analyses were used to investigate the relevance of these microbial ecosystems in the formation of stalactites. Ferric hydroxide produced by acidophilic bacteria and archaea was shown to be deposited onto fungal hyphae, resulting in complex mineralized stalactite-shaped structures. Thus, both archaeal-bacterial and fungal members of the ecosystem were shown to play an active role in the formation of stalactites

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula

The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: A novel insight into chronic wound immunity

The systemic host defence mechanisms, especially innate immunity, in venous leg ulcer patients are poorly investigated. The aim of the current study was to measure Candida albicans killing activity and gene expressions of pro- and anti-inflammatory cytokines and innate immune response regulators, TAM receptors and ligands of peripheral blood mononuclear cells separated from 69 venous leg ulcer patients and 42 control probands. Leg ulcer patients were stratified into responder and non-responder groups on the basis of wound healing properties. No statistical differences were found in Candida killing among controls, responders and non-responders. Circulating blood mononuclear cells of patients overexpress pro-inflammatory (IL-1α, TNFα, CXCL-8) and anti-inflammatory (IL-10) cytokines as well as TAM receptors (Tyro, Axl, MerTK) and their ligands Gas6 and Protein S compared with those of control individuals. IL-1α is notably overexpressed in venous leg ulcer treatment non-responders; in contrast, Axl gene expression is robustly stronger among responders. These markers may be considered as candidates for the prediction of treatment response among venous leg ulcer patients. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd

Antimicrobial Activity of NCR Plant Peptides Strongly Depends on the Test Assays

The symbiosis specific NCR247 and NCR335 cationic plant peptides of Medicago truncatula have been shown to exert antimicrobial activity against a wide range of microbes. However, their antimicrobial efficiency is clearly limited by divalent cations. Here, the antibacterial and antifungal activities of NCR247 and NCR335 peptides were compared to those of the well-characterized peptide antibiotics polymyxin B and the aminoglycoside streptomycin on three model microbes, Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae as representatives of Gram-negative and Gram-positive bacteria as well as eukaryotic fungi. The aim of the study was to assess how the killing efficiency of these peptides depends on various, widely used antimicrobial susceptibility assays. Validated resazurin microdilution assay was used to determine minimal growth inhibitory concentrations in three general test media (MHB, MHBII and low-salt medium LSM). Bactericidal/fungicidal activities were determined by the commonly used drop plate assay. The natural plant peptides showed distinct characteristics, NCR247 had a generally high sensitivity for Ca2+ and Mg2+ in the medium, while NCR335 proved to be a robust and strong antimicrobial agent with comparable efficiency values to polymyxin B. Activity data were confirmed visually, both NCR247 and NCR335 treatments at minimal bactericidal concentrations induced complete disruption of the membranes and provoked cell lysis on all tested microorganisms as observed by scanning electron microscopy

Interaction of cysteine-rich cationic antimicrobial peptides with intact bacteria and model membranes

Antimicrobial peptides are small proteins that exhibit a broad spectrum of antimicrobial activity. Their chemical structure allows them to interact (attach and insert) with membranes. The fine details about this interaction and their mode of action are not fully clarified yet. In order to better understand this mechanism, we have performed in situ atomic force microscopy studies using two types of nodule specific cysteine-rich NCR peptides on Escherichia coli bacteria and on natural purple membrane. On intact bacteria, both NCR247 and NCR335 caused increase in the surface roughness, indicating the damage of the bacterial cell envelope. In case of the tightly packed purple membrane, it is clear that the peptides prefer to disrupt the border of the disks indicating a strong lipid preference of the interaction. These results verify the concept that the first target of NCR peptides is probably the bacterial cell envelope, especially the lipid matrix

Unexplored Arsenals of Legume Peptides With Potential for Their Applications in Medicine and Agriculture

During endosymbiosis, bacteria live intracellularly in the symbiotic organ of their host. The host controls the proliferation of endosymbionts and prevents their spread to other tissues and organs. In Rhizobium-legume symbiosis the major host effectors are secreted nodule-specific cysteine-rich (NCR) peptides, produced exclusively in the symbiotic cells. NCRs have evolved in the Inverted Repeat Lacking Clade (IRLC) of the Leguminosae family. They are secreted peptides that mediate terminal differentiation of the endosymbionts, forming polyploid, non-cultivable cells with increased membrane permeability. NCRs form an extremely large family of peptides, which have four or six conserved cysteines but otherwise highly diverse amino acid sequences, resulting in a wide variety of anionic, neutral and cationic peptides. In vitro, many synthetic NCRs have strong antimicrobial activities against both Gram-negative and Gram-positive bacteria, including the ESKAPE strains and pathogenic fungi. The spectra and minimal bactericidal and anti-fungal concentrations of NCRs differ, indicating that, in addition to their charge, the amino acid composition and sequence also play important roles in their antimicrobial activity. NCRs attack the bacteria and fungi at the cell envelope and membrane as well as intracellularly, forming interactions with multiple essential cellular machineries. NCR-like peptides with similar symbiotic functions as the NCRs also exist in other branches of the Leguminosae family. Thus, legumes provide countless and so far unexplored sources of symbiotic peptides representing an enormous resource of pharmacologically interesting molecules

Impact of Plant Peptides on Symbiotic Nodule Development and Functioning

Ribosomally synthesized peptides have wide ranges of functions in plants being, for example, signal molecules, transporters, alkaloids, or antimicrobial agents. Legumes are an unprecedented rich source of peptides, which are used to control the symbiosis of these plants with the nitrogen-fixing Rhizobium bacteria. Here, we discuss the function and the evolution of these peptides playing an important role in the formation or functioning of the symbiotic organs, the root nodules. We distinguish peptides that can be either cell-autonomous or secreted short-range or long-range signals, carrying messages in or between plant cells or that can act as effectors interacting with the symbiotic bacteria. Peptides are further classified according to the stage of the symbiotic process where they act. Several peptide classes, including RALF, DLV, ENOD40, and others, control Rhizobium infection and the initiation of cell divisions and the formation of nodule primordia. CLE and CEP peptides are implicated in systemic and local control of nodule initiation during autoregulation of nodulation and in response to the nutritional demands of the plant. Still other peptides act at later stages of the symbiosis. The PSK peptide is thought to be involved in the suppression of immunity in nodules and the nodule-specific cysteine-rich, GRP, and SNARP (LEED..PEED) peptide families are essential in the functioning of the nitrogen fixing root nodules. The NCRs and possibly also the GRP and SNARPs are targeted to the endosymbionts and play essential roles in the terminal differentiation of these bacteria