A New Highly Conserved Antibiotic Sensing/Resistance Pathway in Firmicutes Involves an ABC Transporter Interplaying with a Signal Transduction System

Signal transduction systems and ABC transporters often contribute jointly to adaptive bacterial responses to environmental changes. In Bacillus subtilis, three such pairs are involved in responses to antibiotics: BceRSAB, YvcPQRS and YxdJKLM. They are characterized by a histidine kinase belonging to the intramembrane sensing kinase family and by a translocator possessing an unusually large extracytoplasmic loop. It was established here using a phylogenomic approach that systems of this kind are specific but widespread in Firmicutes, where they originated. The present phylogenetic analyses brought to light a highly dynamic evolutionary history involving numerous horizontal gene transfers, duplications and lost events, leading to a great variety of Bce-like repertories in members of this bacterial phylum. Based on these phylogenetic analyses, it was proposed to subdivide the Bce-like modules into six well-defined subfamilies. Functional studies were performed on members of subfamily IV comprising BceRSAB from B. subtilis, the expression of which was found to require the signal transduction system as well as the ABC transporter itself. The present results suggest, for the members of this subfamily, the occurrence of interactions between one component of each partner, the kinase and the corresponding translocator. At functional and/or structural levels, bacitracin dependent expression of bceAB and bacitracin resistance processes require the presence of the BceB translocator loop. Some other members of subfamily IV were also found to participate in bacitracin resistance processes. Taken together our study suggests that this regulatory mechanism might constitute an important common antibiotic resistance mechanism in Firmicutes. [Supplemental material is available online at http://www.genome.org.

Combined systems approaches reveal highly plastic responses to antimicrobial peptide challenge in Escherichia coli

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies

Bacteriocins of Streptococcus pneumoniae and its response to challenges by antimicrobial peptides

Samenvatting voor de leek. Introduction. Biologie is een natuurwetenschap die zich bezighoudt met leven en levende organismen, inclusief hun structuur, functie, groei, oorsprong, ontwikkeling, voortplanting en taxonomie. Omdat Biologie zo‘n buitengewoon breed onderwerp is, wordt het tegenwoordig opgedeeld in verschillende disciplines. Deze onderverdeling is gebaseerd op twee criteria: i) op de biologische organisaties, bijvoorbeeld moleculen, cellen, individuen, populaties en ii) op het specifieke onderwerp dat wordt onderzocht bijvoorbeeld, structuur en functie, groei en ontwikkeling. Op basis van deze criteria kan men een onderverdeling maken in: a) botanie, de plantenstudie, b) zoologie, de dierenstudie, c) microbiologie, de studie naar microscopische organismen, zoals bacteria, d) virologie, de studie naar virussen, e) moleculaire biologie, de studie naar biologische functie op moleculair niveau, f) biochemie, de studie naar chemische reacties die nodig zijn voor de levensvatbaarheid en het functioneren van organismen, g) genetica, de studie naar genen en erfelijkheid en h) moleculaire genetica, de studie naar de structuur en functie van genen op een moleculair niveau. Een molecuul kan worden beschreven als het kleinste deeltje van een stof dat dezelfde chemische en fysieke eigenschappen als de stof zelf. In de 17e eeuw kwam de biologie in een stroomversnelling toen een Nederlander, Antony van Leeuwenhoek, de microscoop verbeterde. Hij wordt beschouwd als de grondlegger van de microbiologie omdat hij de eerste was die ééncellige organismen, zogenaamde microorganismen, onderzocht en beschreef, en is nu bekend als ―de vader van de microbiologie‖. Microorganismen of microben zijn zo klein dat ze niet met het blote oog kunnen worden waargenomen. Ze vormen een diverse groep en bestaan uit bacteria, virussen, schimmels, algen en dieren zoals plankton. Virussen zijn echter niet-levende organismen, omdat ze niet de structuur van een levende cel bezitten en worden daarom gerefereerd als kleine infectieoverdragers. De fundametele bouwsteen van leven is de cel en alle levende organismen bestaan uit één of meer van deze bouwstenen (Fig. 1). Figuur 1 illustreert de verschillen tussen de structuur en componenten van dierencellen, plantencellen en bacteriecellen. De componenten van elke cel bestaan uit moleculen. Over het algemeen kan men een aantal moleculen onderscheiden: 1) organische moleculen zoals proteïnen, carbohydrate, vetzuren, nucleinezuren en 2) niet-organische moleculen zoals water, stikstof, metalen en niet-metalen.

Generic and Specific Adaptive Responses of Streptococcus pneumoniae to Challenge with Three Distinct Antimicrobial Peptides, Bacitracin, LL-37, and Nisin▿ †

To investigate the response of Streptococcus pneumoniae to three distinct antimicrobial peptides (AMPs), bacitracin, nisin, and LL-37, transcriptome analysis of challenged bacteria was performed. Only a limited number of genes were found to be up- or downregulated in all cases. Several of these common highly induced genes were chosen for further analysis, i.e., SP0385-SP0387 (SP0385-0387 herein), SP0912-0913, SP0785-0787, SP1714-1715, and the blp gene cluster. Deletion of these genes in combination with MIC determinations showed that several putative transporters, i.e., SP0785-0787 and SP0912-0913, were indeed involved in resistance to lincomycin and LL-37 and to bacitracin, nisin, and lincomycin, respectively. Mutation of the blp bacteriocin immunity genes resulted in an increased sensitivity to LL-37. Interestingly, a putative ABC transporter (SP1715) protected against bacitracin and Hoechst 33342 but conferred sensitivity to LL-37. A GntR-like regulator, SP1714, was identified as a negative regulator of itself and two of the putative transporters. In conclusion, we show that resistance to three different AMPs in S. pneumoniae is mediated by several putative ABC transporters, some of which have not been associated with antimicrobial resistance in this organism before. In addition, a GntR-like regulator that regulates two of these transporters was identified. Our findings extend the understanding of defense mechanisms of this important human pathogen against antimicrobial compounds and point toward novel proteins, i.e., putative ABC transporters, which can be used as targets for the development of new antimicrobials

Production of a Class II Two-Component Lantibiotic of Streptococcus pneumoniae Using the Class I Nisin Synthetic Machinery and Leader Sequence▿

Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics