The development of tolerance in Pseudomonas putida DOT-T1 to toluene and related highly toxic compounds involves short-and long-term responses. The short-term response is based on an increase in the rigidity of the cell membrane by rapid transformation of the fatty acid cis-9,10-methylene hexadecanoic acid (C17:cyclopropane) to unsaturated 9-cis-hexadecenoic acid (C16:1,9 cis) and subsequent transformation to the trans isomer. The long-term response involves in addition to the changes in fatty acids, alterations in the level of the phospholipid polar head groups: cardiolipin increases and phosphatidylethanolamine decreases. The two alterations lead to increased cell membrane rigidity and should be regarded as physical mechanisms that prevent solvent penetrance. Biochemical mechanisms that decrease the concentration of toluene in the cell membrane also take place and involve: (i) a solvent exclusion system and (ii) metabolic removal of toluene via oxidation. Mutants unable to carry out cis 3 trans isomerization of unsaturated lipids, that exhibit altered cell envelopes because of the lack of the OprL protein, or that are unable to exclude toluene from cell membranes are hypersensitive to toluene.Organic solvents with a logP OW value (logarithm of the partition coefficient of the target compound in a mixture of octanol/ water) between 1.5 and 3 are extremely toxic to microorganisms, a characteristic that has been well documented for toluene (logP OW 2.5) (1-4). De Smet et al. (2) demonstrated that toluene destabilizes the inner membrane of Gram-negative bacteria, causing a transition from a lamellar bilayer state to a hexagonal state, which results in the leakage of proteins, lipids, and ions and disruption of the cell membrane potential (1, 2). The consequent collapse of ATP synthesis together with other lesions lead to cell death.Inoue and Horikoshi (5) isolated a Pseudomonas putida strain able to grow in a double phase system that contained up to 50% (v/v) toluene, despite the fact that this microorganism was not able to use this aromatic as a carbon source. This report was followed by three independent studies that described the isolation of three different P. putida strains that tolerated related organic solvents, e.g. styrene (6), xylenes (7), and toluene (8). The toluene-tolerant isolate, called P. putida DOT-T1, metabolized toluene via the p-cresol pathway (8). The "unexpected" ability of these Pseudomonas strains to tolerate toxic solvents opens new avenues of research into cellular metabolism. In this study, we have explored the molecular basis for solvent tolerance by P. putida DOT-T1. EXPERIMENTAL PROCEDURESBacterial Strains and Culture Conditions-P. putida DOT-T1 is a solvent-tolerant strain (8), whereas P. putida mt-2 is a toluene-sensitive strain (9).Isolation of Toluene-sensitive Tn5 Mutants of P. putida DOT-T1-About 2000 Tn5 transconjugants of P. putida DOT-T1 were obtained after mating this strain with Escherichia coli (pGS9). The suicide plasmid pGS9 bears Tn5, and mutagenesis was carried out a...
Drug discovery from marine natural products has experienced a revival since the beginning of this century. To be successful in this field, rapid dereplication (identification of already known bioactive compounds) is essential in order to assess the chemical novelty of crude extracts and their fractions. Access to the appropriate state-of-the-art analytical instrumentation and to suitable databases is a fundamental requirement in such a task. A brief survey of the most robust LC/UV/MS- and NMR-based approaches employed for marine natural product dereplication is presented alongside a description of the procedures followed to achieve this goal in our research group.
Lantibiotics are ribosomally synthesized and post‐translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d‐amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N‐dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram‐positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.
Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.
Keywords drug design; enzyme inhibition; isothermal titration calorimetry; protein structures; tuberculosis Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately two billion people worldwide, and an estimated nine million of these develop TB each year. [1,2] TB is currently the leading cause of infectious disease mortality in the world by a bacterial pathogen, and claimed an estimated 1.7 million deaths in 2006. [3] As a result of the increasing manifestation of multiple-drug-resistant strains of M. tuberculosis and of the limitations of the current anti-TB therapies, the development of safe and effective new drugs with novel modes of action is urgently needed. [4] Pantothenate (vitamin B 5 ) is the essential precursor to coenzyme A and acyl carrier proteins. The de novo biosynthetic pathway to pantothenate is present in many bacteria, fungi and plants and comprises four enzymes, encoded by panB, panE, panD and panC. [5] Bioinformatics analyses have identified this pathway as a potential target for antimicrobial agents. [6] The absence of each enzyme in mammals further suggests that inhibitors could be selective with a reduced risk of side effects. Crucially, genetic studies have shown that a pantothenate auxotroph of M. tuberculosis defective in the panC and panD genes fails to establish virulence in a mouse model of infection. [7] An attenuated strain of M. tuberculosis that deletes both panCD and the primary attenuating mutations of the bacille Calmette- Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Accession codes:The atomic coordinates and structure factors for the apoenzyme and enzyme-ligand complexes have been deposited in the Protein Data Bank with the following ID codes: 3cov (apo-PS), 3cow (PS-2), 3coy (PS-3) and 3coz (PS-4).
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