DOCSLIB.ORG

Autolytic Mechanism for Spheroplast Formation in Bacillus Cereus and Escherichia Coli RAAM R

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Autolytic Mechanism for Spheroplast Formation in Bacillus Cereus and Escherichia Coli RAAM R JOURNAL OF BACTERIOLOGY, Nov., 1965 Vol. 90, No. 5 Copyright © 1965 American Society for Microbiology Printed in U.S.A. Autolytic Mechanism for Spheroplast Formation in Bacillus cereus and Escherichia coli RAAM R. MOHAN, DONALD P. KRONISH, ROLAND S. PIANOTTI, RAY L. EPSTEIN,' AND BENJAMIN S. SCHWARTZ Department of Microbiology, Warner-Lambert Research Institute, Morris Plains, New Jersey Received for publication 11 June 1965 ABSTRACT MOHAN, RAAM R. (Warner-Lambert Research Institute, Morris Plains, N.J.), DONALD P. KRONISH, ROLAND S. PIANOTTI, RAY L. EPSTEIN, AND BENJAMIN S. SCHWARTZ. Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. J. Bacteriol. 90:1355-1364. 1965.-Spheroplasts of Bacillus cereus strain T and Escherichia coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incuba- tion in 0.05 M tris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 M sodium acetate buffer osmotically stabilized with 1.6 M sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined. B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereus and E. coli indi- cated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopi- melic acid, glutamic acid, and muramic acid. Spheroplast formation has been described in a cereus 130. The material, a protein, was precipi- wide variety of bacterial species (McQuillen, tated by (NH4)2SO4, was inactivated by heat or 1960), some fungi (Bachman and Bonner, 1959), trypsin treatment, and was irreversibly denatured and in at least one instance in higher plants at pH 4.0. In an osmotically stabilized system, (Cocking, 1960). Mitchell and Moyle (1957) showed that, under Methods for the physical, chemical, and enzy- certain conditions in the absence of lysozyme, matic preparation of spheroplasts from both suspensions of Staphylococcus aureus became gram-positive and gram-negative bacteria have osmotically sensitive, presumably by autodiges- been discussed in reviews by Salton (1961) and tion of the cell wall, but they did not examine the WVork (1961). Relatively little information, how- characteristics of the spheroplasts produced or the ever, is available on an autolytic mechanism of optimal conditions required for their production. spheroplast production. Nomura and Hosoda Kato et al. (1962) isolated a lytic factor desig- (1956) isolated an "autolysin" by (NH4)2SO4 nated Li, enzyme from culture supernatant liq- precipitation of autolysates of Bacillus subtilis uids of Flavobacterium sp., which exhibited lytic strain H, which caused lysis of B. subtilis and activity against intact cells and isolated cell walls B. megaterium but did not give rise to stable of S. aureus and Micrococcus lysodeikticus. spherophasts. Dark and Strange (1957) iso- The principal advantage of an autolytic system lated an enzyme from sporulating cells of for spheroplast production and recovery of de- B. cereus which was capable of rapidly and quan- graded cell wall lies in the elimination of any titatively converting cells of the same species to requirements for extraneous supplements, with spheroplasts. A lytic factor induced by ultra- the concomitant contamination of isolates. violet irradiation of B. cereus 569 was reported by The present report describes an autolytic sys- Csuzi and Kramer (1962) to induce lysis in B. tem operative in B. cereus strain T and Escherichia 1 Present address: Department of Bacteriology, coli B, optimal conditions for the production of University of Wisconsin, Madison. spheroplasts, and qualitative characterization of 1355 1356 MOHAN ET AL. J. BACTERIOL. recovered degraded cell-wall material. The pres- suspended in distilled water (100 ml), and disin- ent work was reported in preliminary form by tegrated at 10 C in a Raytheon 10-kc sonic oscilla- Kronish et al. p. 1960). tor for 15 min. The disintegrated cell mass was (Bacteriol. Proc., 63, centrifuged at 10,000 X g for 90 min, and the top white fluffy layer was carefully removed from the MATERIALS AND METHODS pellet. After three additional washings in distilled B. cereus T used in these studies was obtained water at 10,000 X g, the fluffy layer was treated with from H. 0. Halvorson, University of Wisconsin. deoxynuclease and ribonuclease at 40 C for 90 E. coli B was obtained from R. McDonald, Cornell min. The residual pellet was then washed eight University. times in distilled water. All operations except di- Growth of cells. B. cereus was grown in the salts- gestion were carried out at 4 C. The final washed glucose-yeast extract G medium of Stewart and pellet contained white fluffy cell-wall particulate Halvorson (1953). In later experiments, a water- material. In later experiments, equivalent yields dialyzed pancreatic digest of casein (Casitone, of cell wall were obtained by placing the cell mass Difco) replaced yeast extract. Cells were routinely in an Eppenbach colloid mill (model QV-6; grown overnight on a water-bath rotary shaker at Gifford-Wood Co., Hudson, N.Y.) together with 30 C, washed once in sterile distilled water, and 150 ml of distilled water and 90 ml of acid-washed inoculated in fresh medium to give an optical 120-, Superbrite glass beads (Minnesota Mining density of 0.05 at 660 mu (Bausch & Lomb Spec- and Manufacturing Co., St. Paul, Minn.). The tronic-20 colorimeter). colloid mill was run for 5 min at 17 C with a rotor- Cultures were incubated as above and grown to stator gap setting of 0.030 inches, according to the an optical density of 0.60 except where indicated. procedure of Garver and Epstein (1959). Subse- Cells were harvested by centrifugation at 5,000 X quent washing procedures and deoxynuclease and g and were washed once with distilled water. E. ribonuclease treatments were identical to those coli B was grown in the synthetic glucose-salts described above. Isolated cell walls prepared by medium of Davis and Mingioli (1950) at 37 C on a both methods were suspended in Tris or acetate rotary shaker (New Brunswick Scientific Co., New buffers and adjusted to an optical density of ap- Brunswick, N.J.) for 18 hr. A 3% inoculum was proximately 0.40 at 660 mp. Autolysis of the me- made to fresh medium and was incubated at 37 C chanically prepared cell walls from B. cereus and on a rotary shaker. Cells were harvested at an E. coli was determined by measuring the decrease optical density of 0.20 to 0 25 at 660 mu and washed in the optical density of the suspensions during once in 0.2 M NaHCO3. C ell pastes of both organ- incubation at 30 and 37 C, respectively. isms were examined for lytic activity and sphero- Isolation of enzymatically degraded cell wall from plast formation. the spheroplast-forming system. After incubation Lysis and spheroplast formation. B. cereus was of whole cells (until spheroplast formation was suspended in 0.05 M tris(hydroxymethyl)amino- complete), the spheroplast-forming system was methane (Tris) buffer (Sigma Chemical Co., St. centrifuged at 10,000 X g for 30 min to remove Louis, Mo.), pH 7.5, and was adjusted to an optical whole cells, undegraded cell wall, and debris. Cell- density of 0.70 at 660 m,p. Lysis of the cells was re- free supernatant fractions were made 70% with corded during incubation at 30 C by measuring fall respect to ethyl alcohol at -10 C. (Ethyl alcohol in optical density at 660 mA&. Spheroplasts were was cooled to -76 C before addition to prevent formed by suspending the cell paste in Tris buffer local heating and denaturation.) After standing as above, containing 16% (w/v) polyethylene overnight at 4 C, precipitates were centrifuged glycol 4000 (PEG; Union Carbide Chemicals Div., at 5,000 X g and washed twice with cold 70% ethyl New York, N.Y.). Lagerwerff, Ogata, and Eagle alcohol. The resultant pellet was water-soluble (1961) have discussed the osmotic properties of (with the exception of a very slight residue), and PEG. E. coli was suspended in 0.4 M sodium acetate was hydrolyzed in sealed ampoules in 4 N HCl at buffer at pH 6.0 and was incubated at 37 C. Lysis 121 C for 4 hr. Acid was removed by repeated was determined by measuring fall in optical evaporation to dryness prior to chromatography. density at 660 mp. The spheroplast-forming sys- Glucosamine was estimated according to the tem contained 1.6 M sucrose and 1.0 mg/ml of method of Rondle and Morgan (1955). Muramic MgSO4, in addition to sodium acetate as a sta- acid was separated from glucosamine by use of bilizing agent against osmotic lysis. 80% pyridine as the first solvent, followed by n- Mechanical preparation of cell walls. B. cereus butanol-acetic acid-water (68:15:30, v/v) in the and E. coli were grown in 10-liter batches in a New second dimension; its identity was established by Brunswick variable-drive fermentor (New Bruns- comparison with the RF value of an authentic wick Scientific Co.). Cultures were agitated by sample. Amino acid composition of hydrolysates horizontal impeller blades rotating at 250 rev/min was determined by two-dimensional paper chro- and were aerated at 0.2 liters per min through a disc-type sintered stainless-steel sparger.
Load more

Recommended publications
  • Gram-Negative Bacteria; for Instance, Coetzee Spheroplast Formation
    Agric. Biol. Chem., 49 (1), 133-140, 1985 133 Fusion of Spheroplasts and Genetic Recombination of Zymomonasmobilis Hideshi Yanase, Masaru Yasui, Takayuki Miyazaki and KenzoTonomura Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Mozu-ume-machi, Sakai, Osaka 591, Japan Received July 1.8, 1984 Spheroplasts of Zymomonasmobilis (Z-6) were prepared by cultivating it in a hypertonic mediumcontaining penicillin G or glycine at 30°C for more than 6 hr. Thereversion of spheroplasts to a bacillary form was observed at frequencies of 10~2 to 10~3 whenspheroplasts were incubated on hypertonic solid agar plates overlaid with soft agar. Spheroplast fusion was attempted with polyethylene glycol 6000 by crossing two mutants lacking the ability to ferment sugars. Fusants were selected, and obtained at high frequencies. The properties of a stable fusant are discussed. Recently broad host-range plasmids have Media. For the cultivation of Zymomonasstrains, RM and T media were used. RMmediumcontained 2% been transferred into Zymomonasmobilis by glucose, 1.0% yeast extract and 0.2% KH2PO4, pH 6.0. T conjugation.1 ~4) However, transformation medium contained 10% glucose, 1% yeast extract, 1% and transduction have not yet been accom- KH2PO4, 1% (NH4)2SO4 and 0.05% MgSO4-7H2O, pH plished. Besides, cell fusion could be regarded 5.6. For the formation of spheroplasts, S mediumwas used as a useful method for strain improvement. containing 20%sucrose, 2%glucose, 1%yeast extract, There have been manyreports indicating pro- 0.2% KH2PO4 and 0.2% MgSO4à"7H2O, pH 6.0. For the toplast fusion in Gram-positive bacteria such regeneration, R mediumwas used containing 16% sor- as Bacillus^ Staphylococcus,6) Brevibacte- bitol, 2% glucose, 1% yeast extract and 0.2% KH2PO4, pH riurn1) and Streptomyces^ but only a few on Gram-negative bacteria; for instance, Coetzee Spheroplast formation.
    [Show full text]
  • Distinct Single-Cell Morphological Dynamics Under Beta-Lactam Antibiotics
    Molecular Cell Article Distinct Single-Cell Morphological Dynamics under Beta-Lactam Antibiotics Zhizhong Yao,1,3 Daniel Kahne,1,4,* and Roy Kishony2,3,* 1Department of Chemistry and Chemical Biology 2School of Engineering and Applied Sciences Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA 3Department of Systems Biology 4Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA *Correspondence: [email protected] (D.K.), [email protected] (R.K.) http://dx.doi.org/10.1016/j.molcel.2012.09.016 SUMMARY in cell division, elongation, and shape maintenance (Spratt, 1975). It has been proposed that inhibition of crosslink forma- The bacterial cell wall is conserved in prokaryotes, tion by beta-lactams combined with misregulated cell-wall stabilizing cells against osmotic stress. Beta- degradation by PG hydrolases results in the accumulation of lactams inhibit cell-wall synthesis and induce lysis PG defects, which ultimately leads to cell lysis (Chung et al., through a bulge-mediated mechanism; however, 2009). little is known about the formation dynamics and The physical process of PG defect formation and subsequent stability of these bulges. To capture processes of lysis is poorly understood. Previous literature suggested a bulge-mediated process (Chung et al., 2009; Huang et al., different timescales, we developed an imaging 2008), and the reported rates of lysis have been loosely charac- platform combining automated image analysis with terized as slow and fast (de Pedro et al., 2002). However, in the live-cell microscopy at high time resolution. Beta- absence of systematic characterization of bulge-formation lactam killing of Escherichia coli cells proceeded dynamics and their variability across individual cells, it is unclear through four stages: elongation, bulge formation, whether lysis occurs uniformly within isogenic cell populations, bulge stagnation, and lysis.
    [Show full text]
  • The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia Coli Dh5α IRIS LIU, MARTHA LIU, KAREN SHERGILL Microbiology and Immunology, UBC
    Journal of Experimental Microbiology and Immunology (JEMI) Vol. 9:81-85 Copyright © April 2006, M&I, UBC The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia coli DH5α IRIS LIU, MARTHA LIU, KAREN SHERGILL Microbiology and Immunology, UBC Based on the observation that transformation in Escherichia coli is an inefficient process, it was hypothesized that transformation may be hindered by the presence of the outer membrane and the cell wall. To test this hypothesis, cells which lack both a cell wall and most of its outer membrane, termed spheroplasts, were created and transformed with pUC8 plasmid. Spheroplast formation was induced by incubating exponentially growing E. coli DH5α in ampicillin and sucrose. To transform these spheroplasts, the cells were subjected to a modified version of the Hanahan protocol, which involves treating the cells with calcium chloride. The results of this experiment suggest that spheroplasts can transform at 4.9 × 10-4. This is 100 fold more efficient than using the modified Hanahan protocol on wildtype E. coli DH5α cells. ________________________________________________________________ Competence is the ability of a bacterium to take up and physical aspects of the outer membrane hinders foreign DNA from the environment and the DNA uptake. If this is true, then removing the outer recombination or incorporation of this DNA is known membrane should increase transformation efficiency. as transformation (14). Although Escherichia coli is In the past, many groups have created strains of E. not considered to be a naturally competent organism, coli, such as ML-308, which have lost most their cell competence can be developed through various wall and only have remnants of an outer membrane (1, procedures.
    [Show full text]
  • Automated Image Analysis for Quantification of Filamentous Bacteria
    Syddansk Universitet Automated image analysis for quantification of filamentous bacteria Fredborg, M.; Rosenvinge, Flemming Schønning; Spillum, E.; Kroghsbo, S.; Wang, M.; Søndergaard, Teis Esben Published in: B M C Microbiology DOI: 10.1186/s12866-015-0583-5 Publication date: 2015 Document version Publisher's PDF, also known as Version of record Document license CC BY Citation for pulished version (APA): Fredborg, M., Rosenvinge, F. S., Spillum, E., Kroghsbo, S., Wang, M., & Sondergaard, T. E. (2015). Automated image analysis for quantification of filamentous bacteria. B M C Microbiology, 15, [255]. DOI: 10.1186/s12866- 015-0583-5 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Sep. 2018 Fredborg et al. BMC Microbiology (2015) 15:255 DOI 10.1186/s12866-015-0583-5 METHODOLOGY ARTICLE Open Access Automated image analysis for quantification of filamentous bacteria Marlene Fredborg1,4*, Flemming S.
    [Show full text]
  • Final Report of the Lyme Disease Review Panel of the Infectious Diseases Society of America (IDSA)
    Final Report of the Lyme Disease Review Panel of the Infectious Diseases Society of America (IDSA) INTRODUCTION AND PURPOSE In November 2006, the Connecticut Attorney General (CAG), Richard Blumenthal, initiated an antitrust investigation to determine whether the Infectious Diseases Society of America (IDSA) violated antitrust laws in the promulgation of the IDSA’s 2006 Lyme disease guidelines, entitled “The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America” (the 2006 Lyme Guidelines). IDSA maintained that it had developed the 2006 Lyme disease guidelines based on a proper review of the medical/scientifi c studies and evidence by a panel of experts in the prevention, diagnosis, and treatment of Lyme disease. In April 2008, the CAG and the IDSA reached an agreement to end the investigation. Under the Agreement and its attached Action Plan, the 2006 Lyme Guidelines remain in effect, and the Society agreed to convene a Review Panel whose task would be to determine whether or not the 2006 Lyme Guidelines were based on sound medical/scientifi c evidence and whether or not these guidelines required change or revision. The Review Panel was not charged with updating or rewriting the 2006 Lyme Guidelines. Any recommendation for update or revision to the 2006 Lyme Guidelines would be conducted by a separate IDSA group. This document is the Final Report of the Review Panel. REVIEW PANEL MEMBERS Carol J. Baker, MD, Review Panel Chair Baylor College of Medicine Houston, TX William A. Charini, MD Lawrence General Hospital, Lawrence, MA Paul H.
    [Show full text]
  • Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative
    bioRxiv preprint doi: https://doi.org/10.1101/578559; this version posted March 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Spheroplast-mediated carbapenem tolerance in Gram-negative 2 pathogens 3 4 Trevor Cross *1, Brett Ransegnola *1, Jung-Ho Shin 1, Anna Weaver 1, Kathy Fauntleroy 5 2, Mike VanNieuwenhze 3, Lars F. Westblade 2,4# and Tobias Dörr 1# 6 7 1 Weill Institute for Cell and Molecular Biology and Department of Microbiology, Cornell University, Ithaca, 8 NY, USA 9 2 Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA 10 3 Department of Molecular and Cellular Biochemistry and Department of Biology, Indiana University, 11 Bloomington, IN, USA 12 4 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, NY, USA 13 14 *These authors contributed equally to this work 15 #To whom correspondence should be addressed: 16 [email protected] or [email protected] 17 18 Running title: Spheroplast-mediated carbapenem tolerance 19 20 Keywords: antibiotic, carbapenem, carbapenemase, Gram-negative, L- 21 form, meropenem, tolerance 22 23 bioRxiv preprint doi: https://doi.org/10.1101/578559; this version posted March 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 Antibiotic tolerance, the ability to temporarily sustain viability in the presence of 26 bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of 27 antibiotic treatment failure.
    [Show full text]
  • Horizontal Gene Transfers and Cell Fusions in Microbiology, Immunology and Oncology (Review)
    441-465.qxd 20/7/2009 08:23 Ì ™ÂÏ›‰·441 INTERNATIONAL JOURNAL OF ONCOLOGY 35: 441-465, 2009 441 Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review) JOSEPH G. SINKOVICS St. Joseph's Hospital's Cancer Institute Affiliated with the H. L. Moffitt Comprehensive Cancer Center; Departments of Medical Microbiology/Immunology and Molecular Medicine, The University of South Florida College of Medicine, Tampa, FL 33607-6307, USA Received April 17, 2009; Accepted June 4, 2009 DOI: 10.3892/ijo_00000357 Abstract. Evolving young genomes of archaea, prokaryota or immunogenic genetic materials. Naturally formed hybrids and unicellular eukaryota were wide open for the acceptance of dendritic and tumor cells are often tolerogenic, whereas of alien genomic sequences, which they often preserved laboratory products of these unisons may be immunogenic in and vertically transferred to their descendants throughout the hosts of origin. As human breast cancer stem cells are three billion years of evolution. Established complex large induced by a treacherous class of CD8+ T cells to undergo genomes, although seeded with ancestral retroelements, have epithelial to mesenchymal (ETM) transition and to yield to come to regulate strictly their integrity. However, intruding malignant transformation by the omnipresent proto-ocogenes retroelements, especially the descendents of Ty3/Gypsy, (for example, the ras oncogenes), they become defenseless the chromoviruses, continue to find their ways into even the toward oncolytic viruses. Cell fusions and horizontal exchanges most established genomes. The simian and hominoid-Homo of genes are fundamental attributes and inherent characteristics genomes preserved and accommodated a large number of of the living matter.
    [Show full text]
  • Morphological and Ultrastructural Changes in Bacterial Cells As an Indicator of Antibacterial Mechanism of Action
    Cell. Mol. Life Sci. DOI 10.1007/s00018-016-2302-2 Cellular and Molecular Life Sciences REVIEW Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action 1 2 2 T. P. Tim Cushnie • Noe¨lle H. O’Driscoll • Andrew J. Lamb Received: 24 April 2016 / Revised: 21 June 2016 / Accepted: 28 June 2016 Ó Springer International Publishing 2016 Abstract Efforts to reduce the global burden of bacterial highlighting those morphological and ultrastructural chan- disease and contend with escalating bacterial resistance are ges which are consistently induced by agents sharing the spurring innovation in antibacterial drug and biocide same mechanism (e.g. spheroplast formation by peptido- development and related technologies such as photody- glycan synthesis inhibitors) and explaining how changes namic therapy and photochemical disinfection. Elucidation that are induced by multiple antibacterial classes (e.g. fil- of the mechanism of action of these new agents and pro- amentation by DNA synthesis inhibitors, FtsZ disruptors, cesses can greatly facilitate their development, but it is a and other types of agent) can still yield useful mechanistic complex endeavour. One strategy that has been popular for information. Lastly, recommendations are made regarding many years, and which is garnering increasing interest due future study design and execution. to recent technological advances in microscopy and a deeper understanding of the molecular events involved, is Keywords Antibiotic Á Antiseptic Á Disinfectant Á the examination of treated bacteria for changes to their Mode of action Á SEM Á TEM morphology and ultrastructure. In this review, we take a critical look at this approach.
    [Show full text]
  • Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts
    International Journal of Molecular Sciences Review Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts Hiromi Nishida Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; [email protected]; Tel.: +81-766-56-7500 Received: 7 August 2020; Accepted: 25 September 2020; Published: 27 September 2020 Abstract: Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement. Keywords: bacterial cell enlargement; microinjection; protoplasts; spheroplasts; metal ion composition; DNA replication; vacuole formation 1. Introduction Bacteria usually grow asexually, inheriting their DNA by clonal production (cell division). Therefore, these clones have the same genetic information. If clonal production were the only form of inheritance in bacteria, bacteria would display limited genetic variation. However, bacteria are so diverse that more than 99% are thought to be unknown at the species level.
    [Show full text]
  • Sonophotocatalysis Mediated Morphological Transition Modulates Virulence and Antibiotic Resistance in Salmonella Typhimurium
    Electronic Supplementary Material (ESI) for Environmental Science: Water Research & Technology. This journal is © The Royal Society of Chemistry 2020 Electronic Supplementary Information Sonophotocatalysis mediated morphological transition modulates virulence and antibiotic resistance in Salmonella Typhimurium A. P. Habeeb Rahman a, b, Ananyo Jyoti Misra a, b, Swagatika Panda c, Bhaskar Das d, Prachi Bhol a, Priti Sundar Mohanty a, b, Ashok J. Tamhankar b, e, Amrita Mishra b, Cecilia Stålsby Lundborg e, Suraj K. Tripathy a, b * a School of Chemical Technology, Kalinga Institute of Industrial Technology, Bhubaneswar, India b School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, India c ICMR-Regional Medical Research Center, Bhubaneswar, India dDepartment of Biotechnology and Medical Engineering, National Institute of Technology, Raurkela, India eDepartment of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden * Corresponding author: S. K. Tripathy (E-mail: [email protected], Tel: +91-674-2725466) Synthesis of Fe-ZnO NPs: Supplementary Scheme. 1 Flowchart of Fe-ZnO NPs synthesis 8 7 6 5 L m / 4 U F C 0 1 3 g o l STm 2 STm SPI SB300 1 STm GFP SEH 0 0 20 40 60 80 100 120 Time (min) Fig. S1. Efficacy of SPC disinfection against different mutants of S. enterica. STm- antibiotic resistant mutant, STm SPI- Salmonella pathogenicity island induced STm, SB300- virulent strain of wild type S. Typhimurium, STm GFP- Green fluorescence protein tagged mutant, SEH- Hydrogen peroxide resistant mutant of S. Enteritidis. (Bacterial loading ≈ 107 CFU/mL, Fe-ZnO NPs loading = 200 mg/L, Temperature=35±2°C). Error bar indicates the standard deviation of replicates (n=3).
    [Show full text]
  • Optical Microscopy Reveals the Dynamic Nature of B. Pseudomallei Morphology During Β-Lactam Antimicrobial Susceptibility Testing Heather P
    McLaughlin et al. BMC Microbiology (2020) 20:209 https://doi.org/10.1186/s12866-020-01865-w RESEARCH ARTICLE Open Access Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing Heather P. McLaughlin* , Julia Bugrysheva and David Sue Abstract Background: In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans. Results: Here, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin- clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis.
    [Show full text]
  • Liposidomycin, the First Reported Nucleoside Antibiotic Inhibitor of Peptidoglycan Biosynthesis Translocase I
    The Journal of Antibiotics (2019) 72:877–889 https://doi.org/10.1038/s41429-019-0241-5 SPECIAL FEATURE: REVIEW ARTICLE Liposidomycin, the first reported nucleoside antibiotic inhibitor of peptidoglycan biosynthesis translocase I: The discovery of liposidomycin and related compounds with a perspective on their application to new antibiotics Ken-ichi Kimura1 Received: 19 July 2019 / Revised: 17 September 2019 / Accepted: 17 September 2019 / Published online: 4 October 2019 © The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019 Abstract Liposidomycin is a uridyl liponucleoside antibiotic isolated from Streptomyces griseosporeus RK-1061. It was discovered by Isono in 1985, who had previously isolated and developed a related peptidyl nucleoside antibiotic, polyoxin, a specific inhibitor of chitin synthases, as a pesticide. He subsequently isolated liposidomycin, a specific inhibitor of bacterial peptidoglycan biosynthesis from actinomycetes, using a similar approach to the discovery of polyoxin. Liposidomycin has no cytotoxicity against BALB/3T3 cells but has antimicrobial activity against Mycobacterium spp. through inhibition of 1234567890();,: 1234567890();,: MraY (MurX) [phospho-N-acetylmuramoyl-pentapeptide transferase (translocase I, EC 2.7.8.13)]. Since the discovery of liposidomycin, several liposidomycin-type antibiotics, including caprazamycin, A-90289, and muraminomycin, have been reported, and their total synthesis and/or biosynthetic cluster genes have been studied. Most advanced, a semisynthetic compound derived from caprazamycin, CPZEN-45, is being developed as an antituberculosis agent. Translocase I is an interesting and tractable molecular target for new antituberculosis and antibiotic drug discovery against multidrug-resistant bacteria. This review is dedicated to Dr Isono on the occasion of his 88th birthday to recognize his role in the study of nucleoside antibiotics.
    [Show full text]