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  • 1
    Language: English
    In: mBio, 01 November 2018, Vol.9(6), p.e02100-18
    Description: The alphaproteobacterium Agrobacterium tumefaciens is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, Agrobacterium serves as a crucial biotechnological tool for genetic manipulation of plant cells. The presence of hundreds of putative sRNAs in this organism suggests a considerable impact of riboregulation on A. tumefaciens physiology. Here, we characterized the biological function of the sRNA PmaR that controls various processes crucial for growth, motility, and virulence. Among the genes directly targeted by PmaR is ampC coding for a beta-lactamase that confers ampicillin resistance, suggesting that the sRNA is crucial for fitness in the competitive microbial composition of the rhizosphere.Small regulatory RNAs play an important role in the adaptation to changing conditions. Here, we describe a differentially expressed small regulatory RNA (sRNA) that affects various cellular processes in the plant pathogen Agrobacterium tumefaciens. Using a combination of bioinformatic predictions and comparative proteomics, we identified nine targets, most of which are positively regulated by the sRNA. According to these targets, we named the sRNA PmaR for peptidoglycan biosynthesis, motility, and ampicillin resistance regulator. Agrobacterium spp. are long known to be naturally resistant to high ampicillin concentrations, and we can now explain this phenotype by the positive PmaR-mediated regulation of the beta-lactamase gene ampC. Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction in vivo and in vitro. Several riboregulators have been implicated in antibiotic resistance mechanisms, such as uptake and efflux transporters, but PmaR represents the first example of an sRNA that directly controls the expression of an antibiotic resistance gene.
    Keywords: Antibiotic Resistance ; Gene Regulation ; Plant-Microbe Interaction ; Posttranscriptional Control ; Regulatory RNA ; Biology
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 2
    Language: English
    In: Frontiers in molecular biosciences, 2017, Vol.4, pp.9
    Description: The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes and via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that CueR is prone to proteolysis by the AAA proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.
    Keywords: AAA+ Proteases ; Clpap ; Clpxp ; Cuer ; Lon ; Merr Family ; Copper Homoeostasis ; Proteolysis
    ISSN: 2296-889X
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  • 3
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2017, Vol.1520, pp.291-306
    Description: Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.
    Keywords: Mass Spectrometry ; Proteomics ; Ribosome Heterogeneity ; Stress ; Staining and Labeling ; Anti-Bacterial Agents -- Pharmacology ; Bacillus Subtilis -- Metabolism ; Ribosomal Proteins -- Metabolism
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 4
    Language: English
    In: Microbiome, 03 March 2017, Vol.5(1), pp.28
    Description: Bacterial biocatalysts play a key role in our transition to a bio-based, post-petroleum economy. However, the discovery of new biocatalysts is currently limited by our ability to analyze genomic information and our capacity of functionally screening for desired activities. Here, we present a simple workflow that combines functional metaproteomics and metagenomics, which facilitates the unmediated and direct discovery of biocatalysts in environmental samples. To identify the entirety of lipolytic biocatalysts in a soil sample contaminated with used cooking oil, we detected all proteins active against a fluorogenic substrate in sample's metaproteome using a 2D-gel zymogram. Enzymes' primary structures were then deduced by tryptic in-gel digest and mass spectrometry of the active protein spots, searching against a metagenome database created from the same contaminated soil sample. We then expressed one of the novel biocatalysts heterologously in Escherichia coli and obtained proof of lipolytic activity.
    Keywords: Biocatalyst ; Lipase ; Metagenomics ; Metaproteomics ; Zymogram ; Escherichia Coli ; Environmental Restoration and Remediation -- Methods ; Lipase -- Genetics ; Lipid Metabolism -- Genetics ; Metagenomics -- Methods ; Proteomics -- Methods
    E-ISSN: 2049-2618
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  • 5
    In: PROTEOMICS – Clinical Applications, October 2016, Vol.10(9-10), pp.1036-1048
    Description: To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.1002/prca.201600039/abstract Byline: Susanne Engelmann, Michael Hecker, Jennifer Janina Stepanek, Sina Schakermann, Sina Schakermann, Pascal Prochnow, Julia Elisabeth Bandow Keywords: I.sub.B-dependent general stress response; Antibiotic; Sporulation; Trimethoprim Purpose Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C.sub.1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Experimental design Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Results Proteins involved in purine and histidine biosynthesis, the I.sub.B-dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. Conclusions and clinical relevance The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. Article Note: Current address: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands Colour Online: See the article online to view Figs. 2 and 3 in colour. Supporting information: Additional Supporting Information may be found in the online version of this article As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. CAPTION(S): prca1778-sup-0001-Supplementary_file_1 prca1778-sup-0001-Supplementary_file_2 prca1778-sup-0001-Supplementary_file_3 prca1778-sup-0001-Supplementary_file_4
    Keywords: Σ B ‐Dependent General Stress Response ; Antibiotic ; Sporulation ; Trimethoprim
    ISSN: 1862-8346
    E-ISSN: 1862-8354
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  • 6
    Language: English
    In: BBA - Biomembranes, May 2018, Vol.1860(5), pp.1114-1124
    Description: Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.
    Keywords: Antibiotic ; Gram-Positive Bacteria ; Rhodomyrtone ; Staphylococcus ; Membrane Active ; Chemistry
    ISSN: 0005-2736
    E-ISSN: 1879-2642
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  • 7
    Language: English
    In: MicrobiologyOpen, 22 August 2019, pp.e921
    Description: Rhodobacter capsulatus fixes atmospheric nitrogen (N ) by a molybdenum (Mo)-nitrogenase and a Mo-free iron (Fe)-nitrogenase, whose production is induced or repressed by Mo, respectively. At low nanomolar Mo concentrations, both isoenzymes are synthesized and contribute to nitrogen fixation. Here we examined the regulatory interplay of the central transcriptional activators NifA and AnfA by proteome profiling. As expected from earlier studies, synthesis of the structural proteins of Mo-nitrogenase (NifHDK) and Fe-nitrogenase (AnfHDGK) required NifA and AnfA, respectively, both of which depend on the alternative sigma factor RpoN to activate expression of their target genes. Unexpectedly, NifA was found to be essential for the synthesis of Fe-nitrogenase, electron supply to both nitrogenases, biosynthesis of their cofactors, and production of RpoN. Apparently, RpoN is the only NifA-dependent factor required for target gene activation by AnfA, since plasmid-borne rpoN restored anfH transcription in a NifA-deficient strain. However, plasmid-borne rpoN did not restore Fe-nitrogenase activity in this strain. Taken together, NifA requirement for synthesis and activity of both nitrogenases suggests that Fe-nitrogenase functions as a complementary nitrogenase rather than an alternative isoenzyme in R. capsulatus.
    Keywords: Rhodobacter ; Anfa Regulon ; Fe-Nitrogenase ; Mo-Nitrogenase ; Nifa Regulon
    E-ISSN: 2045-8827
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 8
    Language: English
    In: Journal of bacteriology, May 2013, Vol.195(9), pp.1912-9
    Description: The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and 〉2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli.
    Keywords: ATP-Dependent Proteases -- Metabolism ; Escherichia Coli -- Enzymology ; Escherichia Coli Proteins -- Metabolism ; Guanine Nucleotides -- Metabolism ; Lipopolysaccharides -- Biosynthesis
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 9
    Language: English
    In: Journal of bacteriology, March 2011, Vol.193(5), pp.1090-7
    Description: Despite the essential function of lipopolysaccharides (LPS) in Gram-negative bacteria, it is largely unknown how the exact amount of this molecule in the outer membrane is controlled. The first committed step in LPS biosynthesis is catalyzed by the LpxC enzyme. In Escherichia coli, the cellular concentration of LpxC is adjusted by the only essential protease in this organism, the membrane-anchored metalloprotease FtsH. Turnover of E. coli LpxC requires a length- and sequence-specific C-terminal degradation signal. LpxC proteins from Salmonella, Yersinia, and Vibrio species carry similar C-terminal ends and, like the E. coli enzyme, were degraded by FtsH. Although LpxC proteins are highly conserved in Gram-negative bacteria, there are striking differences in their C termini. The Aquifex aeolicus enzyme, which is devoid of the C-terminal extension, was stable in E. coli, whereas LpxC from the alphaproteobacteria Agrobacterium tumefaciens and Rhodobacter capsulatus was degraded by the Lon protease. Proteolysis of the A. tumefaciens protein required the C-terminal end of LpxC. High stability of Pseudomonas aeruginosa LpxC in E. coli and P. aeruginosa suggested that Pseudomonas uses a proteolysis-independent strategy to control its LPS content. The differences in LpxC turnover along with previously reported differences in susceptibility against antimicrobial compounds have important implications for the potential of LpxC as a drug target.
    Keywords: Bacterial Proteins -- Metabolism ; Escherichia Coli -- Metabolism ; Gene Expression Regulation, Bacterial -- Physiology ; Lipopolysaccharides -- Biosynthesis ; Pseudomonas Aeruginosa -- Metabolism ; Salmonella Typhimurium -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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