de Dios, R. | bioRxiv Science

bioRxiv2022

A high-efficiency scar-free genome editing toolkit for Acinetobacter baumannii

Structured synopsisO_ST_ABSBackgroundC_ST_ABSThe current mutagenesis tools for Acinetobacter baumannii leave selection markers or residual sequences behind, or involve tedious counterselection and screening steps. Furthermore, they are usually adapted for model strains, rather than to multidrug resistant (MDR) clinical isolates. ObjectivesTo develop a scar-free genome editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075. MethodsWe prove the efficiency of our adapted genome editing system by deleting the multidrug efflux pumps craA and cmlA5, as well as curing plasmid p1AB5075. We then characterised the antibiotic sensitivity phenotype of the mutants compared to the wild type for chloramphenicol, tobramycin and amikacin by disc diffusion assays and determined their minimum inhibitory concentration for each strain. ResultsWe successfully adapted the genome editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and securing the construction of a mutant within 10 work days. Furthermore, we show that the {Delta}craA has a strong sensitivity to chloramphenicol, tobramycin and amikacin, whereas the {Delta}cmlA5 mutant does not show a significant decrease in viability for the antibiotics tested. On the other hand, the removal of p1AB5075 produced an increased sensitivity to tobramycin and amikacin. ConclusionWe have adapted a highly efficient genome editing tool for A. baumannii and proved that craA has a broader substrate range than previously thought. On the other hand, whereas cmlA5 is annotated as a chloramphenicol efflux pump and is encoded within an aminoglycoside resistance island, it does not provide resistance to any of those compounds.

microbiology↗

bioRxiv2021

The functional differences between paralogous regulators define the control of the General Stress Response in Sphingopyxis granuli TFA

Sphingopyxis granuli TFA is a contaminant degrading alphaproteobacterium that responds to adverse conditions by inducing the General Stress Response (GSR), an adaptive response that controls the transcription of a variety of genes to overcome adverse conditions. The GSR triggered by TFA is driven by two extracytoplasmic function {sigma} factors (ECFs), EcfG1 and EcfG2, whose functional differences have been addressed previously, being EcfG2 the main activator. Upstream in this cascade, NepR anti-{sigma} factors directly inhibit EcfG activity under non-stress conditions, whereas PhyR response regulators sequester the NepR elements upon stress sensing to relieve EcfG inhibition. These elements, which are essential mediators of the GSR regulation, are duplicated in TFA, being NepR1 and NepR2, and PhyR1 and PhyR2. Here, based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each of these paralogs and assessed their contribution to the GSR regulation, dictating its timing and the intensity. We show that different stress signals are differentially integrated into the GSR by PhyR1 and PhyR2, therefore producing different levels of GSR activation. We demonstrate in vitro that both NepR1 and NepR2 bind EcfG1 and EcfG2, although NepR1 produces a more stable interaction than NepR2. Conversely, NepR2 interacts with phosphorylated PhyR1 and PhyR2 more efficiently than NepR1. We propose an integrative model where NepR2 would play a dual negative role: it would directly inhibit the {sigma} factors upon activation of the GSR and it would modulate the GSR activity indirectly by titrating the PhyR regulators. IMPORTANCEIn Alphaproteobacteria, the General Stress Response (GSR) aims at protecting against a variety of stresses. Needing to integrate different signals, its modulation is capital to produce a proportionate response according to the environmental conditions. Individual alphaproteobacterial species have evolved distinct GSR cascades in which the information flow is usually straightforward to ascertain due to the presence of a single copy of at least one of its main regulators (PhyR, NepR and EcfG), restricting the regulatory possibilities. However, Sphingopyxis granuli TFA encodes two paralogs of each regulator, multiplying the possible regulatory interplays. We demonstrate that functional differences between paralogous GSR regulators allow an intrinsic feedback regulation in this pathway. We provide evidence of a NepR anti-{sigma} factor that exerts a dual negative feedback regulation on the GSR by interacting with the EcfG {sigma} factors and with the PhyR regulators. This would attune its output to the actual needs of the cell.

microbiology↗