Abstract H-NS is a histone-like nucleoid-structuring protein that regulates gene expressions, particularly acquired foreign genes, however, little is known about whether H-NS can modulate bacterial susceptibility by regulating its intrinsic genes. The hns-deleted mutant EΔhns, the hns-complemented strain EΔhns/phns and the hns-overexpressed strain E/phns were derivatives of Escherichia coli ATCC 25922, the susceptibility of which were assessed by the broth microdilution method and time-kill curves assays. We found that the MICs for strain EΔhns against gentamicin and amikacin were significantly decreased by 8–16 folds in contrast to E. coli ATCC 25922. Further studies displayed that the absence of hns caused damage to the bacterial outer membrane and increased the expression levels of porin-related genes, such as ompC, ompF, ompG, and ompN, thus obviously enhancing aminoglycosides uptake of strain EΔhns. Meanwhile, hns deletion also led to remarkable inhibition of the efflux pumps activity and decreased expressions of efflux-related genes clbM, acrA, acrB, acrD, and emrE, which reduced the efflux of aminoglycosides. In addition, the activation of glycolysis and electron transport chain, as well as the reduction of Δψ dissipation, could lead to a remarkable increase in proton motive force (PMF), thus further inducing more aminoglycosides uptake by strain EΔhns. Our findings reveal that H-NS regulates the resistance of E. coli to aminoglycosides by influencing its uptake and efflux, which will enrich our understanding of the mechanism by which H-NS modulates bacterial resistance. Keywords: H-NS, aminoglycosides, uptake, efflux, glycolysis, proton motive force 1 Introduction The histone-like nucleoid structuring (H-NS) protein is 137 amino acids in length in Escherichia coli and closely related bacteria and is present at very high levels, with approximately 20,000 to 60,000 molecules per cell ([39]1, [40]2). It is generally believed that the H-NS protein, as a global regulator, preferentially binds to AT-rich DNA by preventing RNA polymerase from accessing or escaping promoters ([41]3), thereby silencing the expression of corresponding genes, notably acquired foreign genes, such as resistance genes acquired by horizontal transfer ([42]4, [43]5). To date, H-NS has received considerable attention in regulating acquired foreign genes, and it has been proved that H-NS protects bacteria and drives their evolution via regulating the expression of foreign DNA ([44]6–[45]8). Meanwhile, some literatures have clarified that H-NS can also modulate genes associated with virulence, stress response, quorum sensing, biosynthesis pathways and cell adhesion by recognizing intrinsically curved DNA ([46]9–[47]11). Up to now, there are a few reports on H-NS regulating multidrug resistance by controlling the inherent genes in the bacterial host. Nishino et al. proposed that H-NS contributed to multidrug resistance E. coli by regulating the expression of multidrug exporter genes such as acrEF and mdtEF ([48]12, [49]13). In 2018, Deveson Lucas team found that H-NS inactivation resulted in an increased resistance to colistin in a clinical isolate of Acinetobacter baumannii ([50]14). Rodgers et al. proved that H-NS modulated antibiotic resistance in Acinetobacter baumannii by governing genes codifying for biofilms and efflux pumps ([51]15). Antibiotic resistance is achieved through a variety of mechanisms, such as alteration or bypass of the drug target, production of antibiotic-modifying enzymes, decreased drug uptake, increased drug efflux, and biofilm formation ([52]16). However, little is known about whether H-NS can influence multidrug resistance by interfering drug uptakes. Aminoglycosides have been one of the important antibiotics for preventing Gram-negative bacteria infection since 1940. Nevertheless, the widespread presence of resistance bacteria has led to a sharp decline in its efficacy. Recently, we occasionally found that the deletion of hns increased the susceptibility of E. coli to many antibiotics, especially aminoglycosides, with their minimal inhibitory concentrations (MICs) decreased by 8–16 folds. The work described below elucidates that H-NS regulates the resistance of E. coli ATCC 25922 to aminoglycosides by influencing its uptake and efflux, which will enrich our understanding of the regulatory mechanism of H-NS on bacterial resistance, and also contribute to the development of new drugs to curb bacterial resistance. 2 Materials and methods 2.1 Bacterial strains, plasmids, and primers Bacteria and plasmids used in this study are listed in [53]Table 1. Escherichia coli ATCC 25922 was obtained from the China Institute of Veterinary Drug Control. Strain EΔhns is a derivative of E. coli ATCC 25922 via the one-step inactivation of chromosomal gene hns ([54]17). The complementary strain EΔhns/phns was constructed as follows: Firstly, the complete open reading frame of hns was amplified by PCR from the genomic DNA of E. coli ATCC 25922 using primers XhoI-hns–F/HindIII-hns-R ([55]Table 2). Thereafter, the expression plasmid pBAD::hns was generated by inserting the target fragment to the vector pBAD (Karsbad, CA, the United States) and then was introduced to EΔhns by electroporation. The overexpressed hns strain E/phns is a derivative of E. coli ATCC 25922 that was introduced of pBAD::hns by electroporation. All strains were cultured in fresh Luria-Bertani (LB) broth (Beijing Land Bridge Technology Co., Ltd.) at 37°C, where strains EΔhns, EΔhns/phns and E/phns were induced by 0.2% L-arabinose ([56]18). Table 1. The MICs of antimicrobial agents against the tested strains (μg/mL). Antimicrobial agents E. coli ATCC 25922 EΔhns EΔhns/phns E/phns Gentamicin 1.000 0.063 0.125 1.000 Amikacin 1.000 0.125 0.250 1.000 Doxycycline 0.500 0.250 0.500 0.500 Tigecycline 0.063 0.031 0.063 0.063 Florfenicol 2.000 1.000 2.000 2.000 Cefotaxime 0.031 0.016 0.016 0.031 Enrofloxacin 0.008 0.002 0.008 0.008 [57]Open in a new tab Strain EΔhns is the derivative of E. coli ATCC 25922 that lacks hns, strain EΔhns/phns is the derivative of EΔhns that carries the recombined plasmid pBAD::hns, while strain E/phns is the derivative of E. coli ATCC 25922 that carries pBAD::hns. Strains EΔhns, EΔhns/phns and E/phns are induced by 0.2% L-arabinose. Table 2. The strains, plasmids and primers used in this study. Strains/plasmids/primers Relevant characteristics References/length