DOCSLIB.ORG

Microcin E492, a Channel-Forming Bacteriocin from Klebsiella Pneumoniae, Induces Apoptosis in Some Human Cell Lines

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microcin E492, a Channel-Forming Bacteriocin from Klebsiella Pneumoniae, Induces Apoptosis in Some Human Cell Lines Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines Claudio Hetz*†, Mari´a Rosa Bono*, Luis Felipe Barros†‡, and Rosalba Lagos*§ *Departamento de Biologı´a,Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile; †Instituto de Ciencias Biome´dicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile; and ‡Centro de Estudios Cientı´ficos,Arturo Prat 514, Valdivia, Chile Communicated by Jorge E. Allende, University of Chile, Santiago, Chile, December 31, 2001 (received for review August 20, 2001) The cytotoxic effect of microcin E492, a low-molecular-mass chan- Apoptosis is a genetically determined form of cell death that nel-forming bacteriocin (7,887 Da) produced by a strain of Kleb- plays a central role during development and homeostasis of siella pneumoniae, was characterized in HeLa cells. At low (5 multicellular organisms (13, 14). Necrotic cell death is usually the ␮g͞ml) and intermediate (10 ␮g͞ml) concentrations, microcin E492 consequence of physical injury and does not involve the active induced biochemical and morphological changes typical of apo- participation of the cell. Apoptosis can be distinguished from ptosis, such as cell shrinkage, DNA fragmentation, extracellular necrosis on the basis of several morphological as well as bio- exposure of phosphatidylserine, caspase activation, and loss of chemical parameters, such as nuclear condensation, loss of cell mitochondrial membrane potential. Treatment with zVAD-fmk, a volume (shrinkage), DNA fragmentation (14), and phosphati- general caspase inhibitor, completely blocked the cytotoxic effect dylserine exposure to the outer face of plasma membrane (15). ␮ ͞ of this bacteriocin. At higher microcin concentrations (>20 g ml) This kind of cell death avoids spillage of intracellular contents in a necrotic phenotype was observed. Induction of apoptosis by contrast to necrotic cell death, typified by cell and organelle microcin E492 was associated with the release of calcium from swelling and membrane disruption, resulting in an inflammatory intracellular stores, probably after microcin-triggered ion channel response (15). The central executioners of apoptosis are a set of formation. Microcin E492 also presented a cytotoxic effect on cysteine proteases that are part of a large protein family known Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a as caspases (16). primary culture of human tonsil endothelial cells, suggesting that Some bacterial toxins share common features with bacterio- there is a specific interaction of the bacteriocin with components cins, such as insertion into or transport across the plasma of the target cell surface. This report describes a bacteriocin that membrane, recognition of the target cell’s membrane, pore has the capacity to induce apoptosis in human cell lines. formation, etc. The known action of bacterial toxins and bacte- riocins has been restricted to eukaryotic cells and bacteria, acteriocins are antimicrobial proteins produced by bacteria respectively, probably because of specific interactions between Bthat are active against closely related species. They are the receptor and the toxin. The only bacteriocin reported to be produced by almost all major lineages of Eubacteria and in lytic for eukaryotic as well as prokaryotic cells is a cytolysin from Archaebacteria (1) and secreted to the extracellular medium Enterococcus faecalis (17). Although it is known that this toxin where they recognize specific receptors on the surface of sen- induces cell lysis, no evidence has been presented as to whether sitive bacterial cells (2). It has been proposed that the primary cytotoxicity is caused by apoptosis or necrosis. role of bacteriocins is to mediate population dynamics within The aim of this work was to study the ability of microcin E492 closely related species (1). Bacteriocins can induce toxicity by a to induce apoptosis. Microcin E492 is a low-molecular-mass variety of mechanisms, most notably the formation of membrane (7,887 Da) channel-forming bacteriocin (18, 19) produced by pores. These compounds have received increasing attention for Klebsiella pneumoniae RYC492, which has a rather broad target their potential use as preservatives in the food industry and as range, being active on strains of the family Enterobacteriaceae antibiotics for clinical usage (3–6). (20). The genetic determinants involved in the production of Bacteria also produce other toxic proteins, released by patho- gens into the host’s body, that are important virulence factors active microcin and its immunity protein have been cloned and causing disease symptoms (tetanus, diphtheria, cholera, etc.). expressed in Escherichia coli (21) and comprise a 13-kb DNA These toxic agents are commonly called bacterial toxins and may fragment encoding for at least 10 genes, necessary for the attack a variety of different cell types (cytotoxins) or specific cell synthesis and export of active microcin (22). Analysis of the types (neurotoxins, hemolysins, etc). Among these bacterial amino acid sequence of microcin E492 indicates that this bac- toxins some can also form membrane pores and kill eukaryotic teriocin does not share structural motif with any other known cells by disrupting the membrane permeability barrier. Lately, protein, and thus, it seems that this microcin belongs to a novel increasing interest has developed in the study of some toxins for class of pore-forming bacteriocins not related to colicin-like their potential therapeutic use as antitumoral drugs. Thus, channel-forming bacteriocin from Gram-negative bacteria or to cytotoxic studies of toxins such as verotoxin 1 (7) and a derivative channel-forming bacteriocins from Gram-positive bacteria (19). of a Pseudomonas exotoxin (8) have demonstrated that these Results presented in this article show that in certain human cell toxins display antitumor activity in vitro and in vivo in ovarian and lines microcin E492 promotes morphological and biochemical breast cancer, respectively. Another aspect that has aroused changes typical of apoptosis. considerable interest is the role of toxin-producing pathogens in promoting apoptosis (9). In contrast to necrosis, apoptosis Abbreviations: AU, antibiotic units; PI, propidium iodide; DiOC6, dihexyloxacarbocyanine; eliminates cells without inducing an inflammatory response, i.e., LDH, lactate dehydrogenase; LFA-1, lymphocyte function-associated antigen 1; TNF-␣, in an immunologically silent manner subverting the normal host tumor necrosis factor ␣. defense responses. Among the pore-forming toxins that induce § ␣ To whom reprint requests should be addressed. E-mail: [email protected]. apoptosis is -toxin, the major cytolysin from Staphylococcus The publication costs of this article were defrayed in part by page charge payment. This aureus (10), aerolysin, produced by Aeromonas hydrophyla (11), article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. and the porin PorB from Neisseria gonorrhoeae (12). §1734 solely to indicate this fact. 2696–2701 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052709699 Downloaded by guest on September 26, 2021 Table 1. Effect of microcin E492 on different human cells types Cell type Cell survival, % Jurkat 4 Ϯ 3 HeLa 56 Ϯ 6 RJ2.2.5 57 Ϯ 11 Ramos 79 Ϯ 19 KG-1 91 Ϯ 1 AMG-3 99 Ϯ 1 Cells were incubated for 24 h in the absence (untreated control) or presence of 680 AU/ml of microcin E492. The percentages of cell survival were deter- mined in respect to the untreated control and are the averages of three independent measurements. Cells were stained with PI, and cell death was determined by flow cytometry analysis. Materials and Methods Isolation, Purification, and Fluorescent Labeling of Microcin E492. Microcin E492 was extracted and purified (20) from the super- natant cultures of E. coli VCS257pJEM15. Microcin activity was estimated and expressed in arbitrary antibiotic units (AU) (23). Normally, 10 ␮g of microcin E492 corresponded to 500 AU. Labeling of microcin with fluorescamine and PAGE were car- ried out as described (21). Cell Lines. The following human cell lines (American Type Culture Collection) were used: HeLa, an epithelial cell line derived from human cervix carcinoma; KG-1, a monocyte- macrophage cell line; RJ2.25, a variant of the Raji B-LCL (24); Jurkat, a T cell derived from acute T cell leukemia, and Ramos, a B cell line originated from Burkitt’s lymphoma. The human endothelial cells from human tonsils (AMG-3) were isolated as described (25). Ramos, Jurkat, RJ2.2.5, KG-1, and AMG-3 cells were maintained in RPMI 1640 medium supplemented with 10% BIOCHEMISTRY FCS and antibiotics. HeLa cell line were maintained in DMEM with 10% FCS and antibiotics. Cytotoxicity and Apoptosis Assays. Cells were seeded at 2 ϫ 104 cells͞well in 96-well plates 24 h before microcin E492 addition, further incubated for the indicated time with microcin E492, ␮ ͞ Fig. 1. Microcin E492 induces cell death with DNA fragmentation in a trypsinized and washed, suspended in PBS, stained with 1 g ml dose-dependent manner. HeLa cells (0.4 ϫ 106 cells in 1 ml) were incubated propidium iodide (PI), and analyzed by using a FACScan during 24 h under different conditions and analyzed by the PI exclusion (Becton Dickinson) with CELL QUEST software. The loss of method. (A) Untreated control. NT, nontreated. (B) Cells incubated with 256 mitochondrial membrane potential was assessed by using 3,3Ј- AU microcin E492. (C) Cells incubated with 1,024 AU microcin E492. Dead cells dihexyloxacarbocyanine [DiOC (3)] (Molecular Probes) as de- permeable to PI are indicated in A–C as M1 and M2 populations. M0 popula- 6 tion represents viable cells, not permeable to PI. mcc, microcin E492. (D) Cells scribed (26). Exposure of phosphatidylserine to the outer leaflet incubated with a preparation from E. coli VCS257np108 with an OD280 equiv- of the plasma membrane was determined by flow cytometry with alent to 10 ␮g. (Inset) A PAGE of microcin E492 (labeled with fluorescamine) annexin V coupled to FITC (Annexin-V-FLUOS staining kit, purified from the microcin-producing strain E.
Load more

Recommended publications
  • Escherichia Coli: Physiological Clues Which Turn on the Synthesis of Antimicrobial Molecules
    veterinary sciences Article Escherichia coli: Physiological Clues Which Turn On the Synthesis of Antimicrobial Molecules Sarah-Jo Paquette 1,2 and Tim Reuter 1,2,* 1 University of Lethbridge, Lethbridge, AB T1K 3M4, Canada; [email protected] 2 Alberta Agriculture and Forestry, Lethbridge, AB T1J 4V6, Canada * Correspondence: [email protected] Received: 5 October 2020; Accepted: 18 November 2020; Published: 21 November 2020 Abstract: Zoonotic pathogens, like Shiga toxin-producing Escherichia coli (STEC) are a food safety and health risk. To battle the increasing emergence of virulent microbes, novel mitigation strategies are needed. One strategy being considered to combat pathogens is antimicrobial compounds produced by microbes, coined microcins. However, effectors for microcin production are poorly understood, particularly in the context of complex physiological responses along the gastro-intestinal tract (GIT). Previously, we identified an E. coli competitor capable of producing a strong diffusible antimicrobial with microcin-associated characteristics. Our objective was to examine how molecule production of this competitor is affected by physiological properties associated with the GIT, namely the effects of carbon source, bile salt concentration and growth phase. Using previously described liquid- and agar-based assays determined that carbon sources do not affect antimicrobial production of E. coli O103F. However, bile salt concentrations affected production significantly, suggesting that E. coli O103F uses cues along the GIT to modulate the expression of antimicrobial production. Furthermore, E. coli O103F produces the molecule during the exponential phase, contrary to most microcins identified to date. The results underscored the importance of experimental design to identify producers of antimicrobials. To detect antimicrobials, conventional microbiological methods can be a starting point, but not the gold standard.
    [Show full text]
  • Peptides Antimicrobiens Des Entérobactériesetude De La Voie De
    Peptides antimicrobiens des entérobactériesEtude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase Sophie Duquesne To cite this version: Sophie Duquesne. Peptides antimicrobiens des entérobactériesEtude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase. Biochimie [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2007. Français. tel-00193192 HAL Id: tel-00193192 https://tel.archives-ouvertes.fr/tel-00193192 Submitted on 1 Dec 2007 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THESE DE DOCTORAT DE L’UNIVERSITE PARIS 6 Spécialité Biochimie Présentée par Sophie DUQUESNE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE PARIS 6 Peptides antimicrobiens des entérobactéries Etude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase Antimicrobial peptides from enterobacteria Maturation process of the antibacterial RNA polymerase inhibitor
    [Show full text]
  • Peptide Bacteriocins – Structure Activity Relationships Hashem Etayash University of Alberta
    Chapman University Chapman University Digital Commons Pharmacy Faculty Articles and Research School of Pharmacy 2015 Peptide Bacteriocins – Structure Activity Relationships Hashem Etayash University of Alberta Sarfuddin Azmi University of Alberta Ramana Dangeti University of Alberta Kamaljit Kaur Chapman University, [email protected] Follow this and additional works at: http://digitalcommons.chapman.edu/pharmacy_articles Recommended Citation Etayash H., Azmi S., Dangeti, R., Kaur K., 2015. Peptide Bacteriocins – Structure Activity Relationships. Current Topics in Medicinal Chemistry 15. DOI: 10.2174/1568026615666150812121103 This Article is brought to you for free and open access by the School of Pharmacy at Chapman University Digital Commons. It has been accepted for inclusion in Pharmacy Faculty Articles and Research by an authorized administrator of Chapman University Digital Commons. For more information, please contact [email protected]. Peptide Bacteriocins – Structure Activity Relationships Comments This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Current Topics in Medicinal Chemistry, volume 15, in 2015 following peer review. The definitive publisher-authenticated version will be available online at DOI: 10.2174/1568026615666150812121103. Copyright Bentham Science This article is available at Chapman University Digital Commons: http://digitalcommons.chapman.edu/pharmacy_articles/201 Peptide Bacteriocins – Structure Activity Relationships Hashem Etayash1,2, Sarfuddin Azmi1, Ramana Dangeti1,
    [Show full text]
  • Structure and Mode of Action of Microcin 7, an Antibacterial Peptide Produced by Escherichia Colit JOSE F
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1985, p. 791-797 Vol. 27, No. 5 00664804/85/050791-07$02.00/0 Copyright X) 1985, American Society for Microbiology Structure and Mode of Action of Microcin 7, an Antibacterial Peptide Produced by Escherichia colit JOSE F. GARCIA-BUSTOS,lt* NIEVES PEZZI,2 AND ENRIQUE MENDEZ3 Instituto de Investigaciones Biomedicas del C.S.I.C., 28029 Madrid,1 Antibioticos S.A., 28015 Madrid,2 and Servicio de Endocrinologia, Centro Ramon y Cajal, 28034 Madrid,3 Spain Received 6 December 1984/Accepted 5 February 1985 Microcin 7 is a small peptide produced and excreted to the culture medium by stationary-phase Escherichia coli cells harboring the pMccC7 plasmid (formerly named pRYC7). This peptide inhibited the growth of the enterobacteria phylogenetically closer to E. coli, apparently by blocking protein biosynthesis. The molecule was degraded with trypsin, and the resulting fragments were purified and sequenced. The results show that microcin 7 is a linear heptapeptide blocked at both ends. Microcins are dialyzable (i.e., low-molecular-weight) an- the peptide (hence the first structural information on a tibacterial agents originally found in bacterial isolates from microcin), the spectrum of its antimicrobial activity, and the the feces of newborn infants (1). Most of the microcins seem first data on its mode of action. to be peptides, and, unlike the classical bacteriocins, they are not inducible by agents that trigger the SOS system for MATERIALS AND METHODS DNA repair (for a review, see reference 4). The synthesis of microcins is not lethal for the producer Trypsin treated with diphenylcarbamyl chloride, carboxy- cells, and it requires the presence of plasmids specific for peptidase Y, phenylthiohydantoin amino acids, and Poly- each type of microcin (3, 21).
    [Show full text]
  • Microcins Mediate Competition Among Enterobacteriaceae in the Inflamed Gut
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Nature. Manuscript Author Author manuscript; Manuscript Author available in PMC 2017 April 30. Published in final edited form as: Nature. 2016 December 08; 540(7632): 280–283. doi:10.1038/nature20557. Microcins mediate competition among Enterobacteriaceae in the inflamed gut Martina Sassone-Corsi1,2, Sean-Paul Nuccio1,2, Henry Liu1, Dulcemaria Hernandez1, Christine T. Vu1, Amy A. Takahashi1, Robert A. Edwards1,3, and Manuela Raffatellu1,2,* 1Department of Microbiology, University of California Irvine, Irvine, CA 92697, USA 2Institute for Immunology, University of California Irvine, Irvine, CA 92697, USA 3Department of Pathology and Laboratory Medicine, University of California, Irvine, CA 92697, USA SUMMARY The Enterobacteriaceae are Gram-negative bacteria and include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine’s microbiota1, some of these organisms can bloom in the inflamed gut2–5; indeed, expansion of enterobacteria is a hallmark of microbial imbalance known as “dysbiosis”6. Microcins are small secreted proteins that possess antimicrobial activity in vitro7,8, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limited growth of competitors in the inflamed intestine, including commensal E. coli, adherent-invasive E. coli, and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild- type, microcin-producing EcN to mice previously infected with S.
    [Show full text]
  • Investigation of Microcin 24 for Applications in Food Safety
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 Investigation of microcin 24 for applications in food safety, mechanism of resistance and effect on activity after site-directed mutagenesis Timothy Steven Frana Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons, and the Molecular Biology Commons Recommended Citation Frana, Timothy Steven, "Investigation of microcin 24 for applications in food safety, mechanism of resistance and effect on activity after site-directed mutagenesis " (2004). Retrospective Theses and Dissertations. 776. https://lib.dr.iastate.edu/rtd/776 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigation of microcin 24 for applications in food safety, mechanism of resistance and effect on activity after site-directed mutagenesis by Timothy Steven Frana A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Microbiology (Preventive Medicine) Program of Study Committee: Ronald W. Griffith, Major Professor Steven Carlson Michael Apley Gregory Phillips James Roth Michael Wannemuehler Iowa State University Ames, Iowa 2004 UMI Number: 3136309 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction.
    [Show full text]
  • Qt6fd707vj.Pdf
    UC San Diego UC San Diego Previously Published Works Title Membrane Porters of ATP-Binding Cassette Transport Systems Are Polyphyletic Permalink https://escholarship.org/uc/item/6fd707vj Journal Journal of Membrane Biology, 231(1) ISSN 1432-1424 Authors Wang, Bin Dukarevich, Maxim Sun, Eric I. et al. Publication Date 2009-09-01 DOI 10.1007/s00232-009-9200-6 Peer reviewed eScholarship.org Powered by the California Digital Library University of California J Membrane Biol (2009) 231:1–10 DOI 10.1007/s00232-009-9200-6 TOPICAL REVIEW Membrane Porters of ATP-Binding Cassette Transport Systems Are Polyphyletic Bin Wang • Maxim Dukarevich • Eric I. Sun • Ming Ren Yen • Milton H. Saier Jr. Received: 20 February 2009 / Accepted: 31 August 2009 / Published online: 6 October 2009 Ó The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The ATP-binding cassette (ABC) superfamily Introduction consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP Over the past 20 years, our laboratory has identified more hydrolyzing constituents, which are monophyletic. The than 600 families of established and putative transport evolutionary origins of the transmembrane porter proteins/ systems (Busch and Saier 2002; Saier 2000), which are domains are not known. Using five distinct computer pro- described and tabulated in the Transporter Classification grams, we here provide convincing statistical data sug- Database (TCDB http://www.tcdb.org) (Saier et al. 2006; gesting that the transmembrane domains of ABC exporters Saier et al. 2009). These families have been defined on the are polyphyletic, having arisen at least three times inde- basis of homology of the transmembrane constituents that pendently.
    [Show full text]
  • Effects of Spaceflight and Simulated Microgravity on Microbial Growth and Secondary Metabolism Bing Huang1†, Dian-Geng Li1†, Ying Huang2* and Chang-Ting Liu1*
    Huang et al. Military Medical Research (2018) 5:18 https://doi.org/10.1186/s40779-018-0162-9 REVIEW Open Access Effects of spaceflight and simulated microgravity on microbial growth and secondary metabolism Bing Huang1†, Dian-Geng Li1†, Ying Huang2* and Chang-Ting Liu1* Abstract Spaceflight and ground-based microgravity analog experiments have suggested that microgravity can affect microbial growth and metabolism. Although the effects of microgravity and its analogs on microorganisms have been studied for more than 50 years, plausible conflicting and diverse results have frequently been reported in different experiments, especially regarding microbial growth and secondary metabolism. Until now, only the responses of a few typical microbes to microgravity have been investigated; systematic studies of the genetic and phenotypic responses of these microorganisms to microgravity in space are still insufficient due to technological and logistical hurdles. The use of different test strains and secondary metabolites in these studies appears to have caused diverse and conflicting results. Moreover, subtle changes in the extracellular microenvironments around microbial cells play a key role in the diverse responses of microbial growth and secondary metabolisms. Therefore, “indirect” effects represent a reasonable pathway to explain the occurrence of these phenomena in microorganisms. This review summarizes current knowledge on the changes in microbial growth and secondary metabolism in response to spaceflight and its analogs and discusses the diverse and conflicting results. In addition, recommendations are given for future studies on the effects of microgravity in space on microbial growth and secondary metabolism. Keywords: Microbial growth, Secondary metabolism, Spaceflight, Microgravity, Simulated microgravity, Microgravity analogs Background [21–23], and microbial mutations and relation to adapta- Microbes are highly evolved [1] and can survive in tion to LSMMG [24].
    [Show full text]
  • Protective Action of Ppgpp in Microcin J25-Sensitive Strainsᰔ Marı´A Fernanda Pomares,† Paula A
    JOURNAL OF BACTERIOLOGY, June 2008, p. 4328–4334 Vol. 190, No. 12 0021-9193/08/$08.00ϩ0 doi:10.1128/JB.00183-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Protective Action of ppGpp in Microcin J25-Sensitive Strainsᰔ Marı´a Fernanda Pomares,† Paula A. Vincent,† Ricardo N. Farı´as, and Rau´l A. Salomo´n* Departamento de Bioquı´mica de la Nutricio´n, Instituto Superior de Investigaciones Biolo´gicas (Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas-Universidad Nacional de Tucuma´n) and Instituto de Quı´mica Biolo´gica “Dr Bernabe´ Bloj”, Chacabuco 461, 4000 San Miguel de Tucuma´n, Tucuma´n, Argentina Received 6 February 2008/Accepted 2 April 2008 As Escherichia coli strains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3؅,5؅-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level. Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, tide substrates reach the catalytic site. Thus, MccJ25 would Downloaded from antibacterial peptide produced by Escherichia coli (32).
    [Show full text]
  • Role of a Microcin-C–Like Biosynthetic Gene Cluster in Allelopathic Interactions in Marine Synechococcus
    Role of a Microcin-C–like biosynthetic gene cluster in allelopathic interactions in marine Synechococcus Javier Paz-Yepesa,b, Bianca Brahamshaa,1, and Brian Palenika,1 aMarine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202; and bInstitut de Biologie de I’Ecole Normale Supérieure, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, 75230 Paris, France Edited* by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 6, 2013 (received for review April 2, 2013) Competition between phytoplankton species for nutrients and in coastal environments. Their discovery in 1979 (19, 20) soon light has been studied for many years, but allelopathic interactions led to a realization of their important contribution to primary between them have been more difficult to characterize. We used productivity (21, 22). It has also become clear that there is sig- liquid and plate assays to determine whether these interactions nificant diversity in the group (21), including distinct genetic occur between marine unicellular cyanobacteria of the genus Syn- clusters with distinct distributions in the water column (23–27), echococcus. We have found a clear growth impairment of Syne- implying the presence of distinct species with different ecological chococcus sp. CC9311 and Synechococcus sp. WH8102 when they niches. One might wonder whether allelopathic interactions are are cultured in the presence of Synechococcus sp. CC9605. The involved in this differentiation. In fact, antibiotic interactions genome of CC9605 contains a region showing homology to genes within microbial communities have been proposed as an effective of the Escherichia coli Microcin C (McC) biosynthetic pathway.
    [Show full text]
  • Transporter Genes in Biosynthetic Gene Clusters Predict Metabolite Characteristics and Siderophore Activity
    Downloaded from genome.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity Alexander Crits-Christoph1,2, Nicholas Bhattacharya3, Matthew R. Olm4, Yun S. Song5,6,9, and Jillian F. Banfield2,4,7,8,9✢ ✢Corresponding author: [email protected] 1Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA 2Innovative Genomics Institute, Berkeley, CA, 94704, USA 3Department of Mathematics, University of California, Berkeley, CA, USA 4Department of Microbiology and Immunology, Stanford University, CA, USA 5Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA, USA 6Department of Statistics, University of California, Berkeley, CA, USA 7Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, USA 8Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 9Chan Zuckerberg Biohub, San Francisco, CA, USA Running title: Transporter genes in biosynthetic gene clusters Keywords: Transporters, specialized metabolism, siderophore 1 Downloaded from genome.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites, and are often genomically co-localized with BGCs. Here we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors.
    [Show full text]
  • Transporter Genes in Biosynthetic Gene Clusters Predict Metabolite Characteristics and Siderophore Activity
    Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Research Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity Alexander Crits-Christoph,1,2 Nicholas Bhattacharya,3 Matthew R. Olm,4 Yun S. Song,5,6,7 and Jillian F. Banfield2,4,7,8,9 1Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA; 2Innovative Genomics Institute, Berkeley, California 94720, USA; 3Department of Mathematics, University of California, Berkeley, California 94720, USA; 4Department of Microbiology and Immunology, Stanford University, California 94305, USA; 5Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, California 94720, USA; 6Department of Statistics, University of California, Berkeley, California 94720, USA; 7Chan Zuckerberg Biohub, San Francisco, California 94158, USA; 8Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720, USA; 9Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this rea- son, genes for transport across cellular membranes are essential for the production of specialized metabolites and are often genomically colocalized
    [Show full text]