Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi

Background This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. Methods R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. Results R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. Conclusions Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

PdMFS1 Transporter Contributes to Penicilliun digitatum Fungicide Resistance and Fungal Virulence during Citrus Fruit Infection

A new Penicillium digitatum major facilitator superfamily (MFS) transporter (PdMFS1) was identified and functionally characterized in order to shed more light on the mechanisms underlying fungicide resistance. PdMFS1 can play an important role in the intensification of resistance to fungicides normally used in P. digitatum postharvest treatments. In the PdMFS1 disrupted mutants, a slight effect in response to chemical fungicides was observed, but fungicide sensitivity was highly affected in the overexpression mutants which became resistant to wide range of chemical fungicides. Moreover, P. digitatum knock-out mutants exhibited a lower rate of fungal virulence when infected oranges were stored at 20 °C. Disease symptoms were higher in the PdMFS1 overexpression mutants coming from the low-virulent P. digitatum parental strain. In addition, the gene expression analysis showed an induction of PdMFS1 transcription in all overexpression mutants regardless from which progenitor came from, and four-time intensification of the parental wild type strain during citrus infection reinforcing PdMFS1 role in fungal virulence. The P. digitatum MFS transporter PdMFS1 contributes not only to the acquisition of wide range of fungicide resistance but also in fungal virulence during citrus infection.

Genome Sequence of a Staphylococcus xylosus Clinical Isolate, Strain SMA0341-04 (UGA5), from Siaya County Referral Hospital in Siaya, Kenya

We report here the genome sequence of a Staphylococcus xylosus clinical isolate, strain SMA0341-04 (UGA5), which contains one chromosome and at least one plasmid. Notably, strain SMA0341-04 (UGA5) contains the tetracycline efflux major facilitator superfamily (MFS) transporter (tetK) gene.

Draft Genome Sequences of Two Staphylococcus warneri Clinical Isolates, Strains SMA0023-04 (UGA3) and SMA0670-05 (UGA28), from Siaya County Referral Hospital, Siaya, Kenya

We report the complete draft genome sequences of two Staphylococcus warneri clinical isolates, strains SMA0023-04 (UGA3) and SMA0670-05 (UGA28), each of which contains one chromosome and at least one plasmid. Isolate SMA0023-04 (UGA3) contains tetracycline efflux major facilitator superfamily (MFS) transporter (tetK), macrolide resistance (msrC and mphC), and beta-lactamase (blaZ) genes on its plasmids.

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake bySphingobiumsp. Strain SYK-6

ABSTRACTThe microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. InSphingobiumsp. strain SYK-6, most of the enzyme genes involved in 5,5′-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (ddvK) and SLG_07780 (ddvR), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. AddvKmutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVAO-demethylase oxygenase component gene (ligXa), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlledligXatranscriptional regulatory system. ASphingobium japonicumUT26S transformant expressingddvKacquired DDVA uptake capacity, indicating thatddvKencodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects ofddvKoverexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (ligI) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of addvK-expression plasmid into aligImutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.IMPORTANCEThe bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5′-dehydrodivanillate, a lignin-derived biphenyl compound, inSphingobiumsp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5′-dehydrodivanillate.