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David K Karig, Seneca Bessling, Peter Thielen, Sherry Zhang, Joshua Wolfe
Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying...
April 2017: Journal of the Royal Society, Interface
Dana Gebhart, Steven R Williams, Dean Scholl
SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C2 and C3 and that a mutation in a putative second tailspike, gp47, eliminates this specificity...
March 10, 2017: Virology
Helena Turano, Fernando Gomes, Gesiele A Barros-Carvalho, Ralf Lopes, Louise Cerdeira, Luis E S Netto, Ana C Gales, Nilton Lincopan
A novel transposon belonging to the Tn3-like family was identified on the chromosome of a commensal strain of Pseudomonas aeruginosa ST2343. Tn6350 is 7,367 bp in size, harboring eight ORFs, an ATPase (IS481 family), a transposase (DDE catalytic type), a Tn3 resolvase, three hypothetical proteins, and genes encoding the new pyocin S8 with their immunity protein. We show that pyocin S8 displays activity against carbapenemase-producing P. aeruginosa, including IMP-1, SPM-1, VIM-1, GES-5 and KPC-2 producers.
February 27, 2017: Antimicrobial Agents and Chemotherapy
Maarten G K Ghequire, Lieselore Kemland, Ernesto Anoz-Carbonell, Susan K Buchanan, René De Mot
Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin's activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module...
February 21, 2017: MBio
Jiale Ma, Zihao Pan, Jinhu Huang, Min Sun, Chengping Lu, Huochun Yao
The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae...
January 6, 2017: Virulence
Fei Chen, Gukui Chen, Yiwei Liu, Yongxin Jin, Zhihui Cheng, Yang Liu, Liang Yang, Shouguang Jin, Weihui Wu
Bacterial oligoribonuclease (Orn) is a conserved 3'-to-5' exonuclease. In Pseudomonas aeruginosa, it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis of P. aeruginosa, indicating a role of Orn in the bacterial response to environmental stimuli...
March 2017: Antimicrobial Agents and Chemotherapy
Grace Lee, Urmi Chakraborty, Dana Gebhart, Gregory R Govoni, Z Hong Zhou, Dean Scholl
UNLABELLED: Listeria monocytogenes is a significant foodborne human pathogen that can cause severe disease in certain high-risk individuals. L. monocytogenes is known to produce high-molecular-weight, phage tail-like bacteriocins, or "monocins," upon induction of the SOS system. In this work, we purified and characterized monocins and found them to be a new class of F-type bacteriocins. The L. monocytogenes monocin genetic locus was cloned and expressed in Bacillus subtilis, producing specifically targeted bactericidal particles...
October 15, 2016: Journal of Bacteriology
Laura C McCaughey, Neil D Ritchie, Gillian R Douce, Thomas J Evans, Daniel Walker
Protein antibiotics, known as bacteriocins, are widely produced by bacteria for intraspecies competition. The potency and targeted action of bacteriocins suggests that they could be developed into clinically useful antibiotics against highly drug resistant Gram-negative pathogens for which there are few therapeutic options. Here we show that Pseudomonas aeruginosa specific bacteriocins, known as pyocins, show strong efficacy in a murine model of P. aeruginosa lung infection, with the concentration of pyocin S5 required to afford protection from a lethal infection at least 100-fold lower than the most commonly used inhaled antibiotic tobramycin...
July 22, 2016: Scientific Reports
Alexander Klein, Justyna Aleksandra Wojdyla, Amar Joshi, Inokentijs Josts, Laura C McCaughey, Nicholas G Housden, Renata Kaminska, Olwyn Byron, Daniel Walker, Colin Kleanthous
Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins...
September 15, 2016: Biochemical Journal
Laura C McCaughey, Inokentijs Josts, Rhys Grinter, Paul White, Olwyn Byron, Nicholas P Tucker, Jacqueline M Matthews, Colin Kleanthous, Cynthia B Whitchurch, Daniel Walker
Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P...
August 1, 2016: Biochemical Journal
Nicholas M I Taylor, Nikolai S Prokhorov, Ricardo C Guerrero-Ferreira, Mikhail M Shneider, Christopher Browning, Kenneth N Goldie, Henning Stahlberg, Petr G Leiman
Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail...
May 19, 2016: Nature
Didier Hocquet, Marie Petitjean, Laurence Rohmer, Benoît Valot, Hemantha D Kulasekara, Elodie Bedel, Xavier Bertrand, Patrick Plésiat, Thilo Köhler, Alix Pantel, Michael A Jacobs, Lucas R Hoffman, Samuel I Miller
When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility...
October 2016: Environmental Microbiology
Lynne Turnbull, Masanori Toyofuku, Amelia L Hynen, Masaharu Kurosawa, Gabriella Pessi, Nicola K Petty, Sarah R Osvath, Gerardo Cárcamo-Oyarce, Erin S Gloag, Raz Shimoni, Ulrich Omasits, Satoshi Ito, Xinhui Yap, Leigh G Monahan, Rosalia Cavaliere, Christian H Ahrens, Ian G Charles, Nobuhiko Nomura, Leo Eberl, Cynthia B Whitchurch
Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs...
2016: Nature Communications
R Fredrik Inglis, Pauline Scanlan, Angus Buckling
The evolution of bacterial resistance to conventional antimicrobials is a widely documented phenomenon with gravely important consequences for public health. However, bacteria also produce a vast repertoire of natural antimicrobials, presumably in order to kill competing species. Bacteriocins are a common class of protein-based antimicrobials that have been shown to have an important role in the ecology and evolution of bacterial communities. Relative to the evolution of antibiotic resistance, little is known about how novel resistance to these toxic compounds evolves...
August 2016: ISME Journal
Jozef Dingemans, Maarten G K Ghequire, Michael Craggs, René De Mot, Pierre Cornelis
S-type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor-binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore-forming activity. Pyocins S2, S3, S4, and S5 recognize TonB-dependent ferri-siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P...
June 2016: MicrobiologyOpen
Dongping Wang, Jun Myoung Yu, Robert J Dorosky, Leland S Pierson, Elizabeth A Pierson
Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30-84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surface-attached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30-84 biofilm matrix is demonstrated...
2016: PloS One
Agustina Godino, Analía Príncipe, Sonia Fischer
Pseudomonas fluorescens SF39a is a plant-growth-promoting bacterium isolated from wheat rhizosphere. In this report, we demonstrate that this native strain secretes bacteriocins that inhibit growth of phytopathogenic strains of the genera Pseudomonas and Xanthomonas. An S-type pyocin gene was detected in the genome of strain SF39a and named pys. A non-polar pys::Km mutant was constructed. The bacteriocin production was impaired in this mutant. To identify genes involved in bacteriocin regulation, random transposon mutagenesis was carried out...
April 2016: Research in Microbiology
Sehar Afshan Naz, Nusrat Jabeen, Muhammad Sohail, Sheikh Ajaz Rasool
Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da...
October 2015: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
Geetika S Choudhary, Xiangyu Yao, Jing Wang, Bo Peng, Rebecca A Bader, Dacheng Ren
Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin...
November 30, 2015: Scientific Reports
Tagbo H R Niepa, Laura M Snepenger, Hao Wang, Shiril Sivan, Jeremy L Gilbert, Marcus B Jones, Dacheng Ren
Recently, we reported synergistic effects between 70 μA/cm(2) direct current and tobramycin in killing Pseudomonas aeruginosa PAO1 persister cells, a phenomenon we named electrochemical control of persister cells (ECCP; Niepa et al. Biomaterials 33: 7356-7365, 2012). To understand the mechanism of ECCP, the effects of electrochemical treatments mediated via stainless steel 304 and carbon electrodes on P. aeruginosa PAO1 were systematically compared using complementary approaches in this study. Electron microscopic analysis revealed that μA/cm(2) level direct current (DC) caused substantial changes in the structure and membrane integrity of P...
January 2016: Biomaterials
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