In the low-G؉C-content Gram-positive bacteria, resistance to antimicrobial peptides is often mediated by so-called resistance modules. These consist of a two-component system and an ATP-binding cassette transporter and are characterized by an unusual mode of signal transduction where the transporter acts as a sensor of antimicrobial peptides, because the histidine kinase alone cannot detect the substrates directly. Thus, the transporters fulfill a dual function as sensors and detoxification systems to confer resistance, but the mechanistic details of these processes are unknown. The paradigm and best-understood example for this is the BceRS-BceAB module of Bacillus subtilis, which mediates resistance to bacitracin, mersacidin, and actagardine. Using a random mutagenesis approach, we here show that mutations that affect specific functions of the transporter BceAB are primarily found in the C-terminal region of the permease, BceB, particularly in the eighth transmembrane helix. Further, we show that while signaling and resistance are functionally interconnected, several mutations could be identified that strongly affected one activity of the transporter but had only minor effects on the other. Thus, a partial genetic separation of the two properties could be achieved by single amino acid replacements, providing first insights into the signaling mechanism of these unusual modules.
Abstract:In a proof of concept study aimed at showing that metabolites in bread wheat (Triticum aestivum L. ssp. aestivum), phloem exudates have potential as biochemical markers for cultivar discrimination, Argentinean cultivars from three quality groups (groups 1, 2, and 3 of high, intermediate, and low quality, respectively) were grown under two nitrogen (N) availabilities and analysed for metabolic profile by electrospray ionisation mass spectrometry. Data as signal strengths of mass/charge (m/z) values binned to a resolution of 0.2 Daltons were subjected to principal component analysis and orthogonal projections to latent structures discriminant analysis. Certain bins were influential in discriminating groups taken in pairs and some were involved in separating all three groups. In high N availability, group 3 cultivars clustered away from the other cultivars, while group 1 cultivars clustered tightly together; group 2 cultivars were more scattered between group 1 and group 3 cultivars. In low N availability, the cultivars were not clustered as tightly; nonetheless, group 1 cultivars tended to cluster together and mainly separated from those of group 2. m/z values also showed potential for discrimination between N availability. In conclusion, phloem exudate metabolic profiles could provide biochemical markers for selection during breeding and for discerning the effects of N fertiliser application.
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