The Stringent Response Promotes Antibiotic Resistance Dissemination by Regulating Integron Integrase Expression in Biofilms

Abstract

Class 1 integrons are genetic systems that enable bacteria to capture and express gene cassettes. These integrons, when isolated in clinical contexts, most often carry antibiotic resistance gene cassettes. They play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. The key element of integrons is the integrase, which allows gene cassettes to be acquired and shuffled. Planktonic culture experiments have shown that integrase expression is regulated by the bacterial SOS response. In natural settings, however, bacteria generally live in biofilms, which are characterized by strong antibiotic resilience and by increased expression of stress-related genes. Here, we report that under biofilm conditions, the stringent response, which is induced upon starvation, (i) increases basal integrase and SOS regulon gene expression via induction of the SOS response and (ii) exerts biofilm-specific regulation of the integrase via the Lon protease. This indicates that biofilm environments favor integron-mediated acquisition of antibiotic resistance and other adaptive functions encoded by gene cassettes.

Importance: Multidrug-resistant bacteria are becoming a worldwide health problem. Integrons are bacterial genetic platforms that allow the bacteria to capture and express gene cassettes. In clinical settings, integrons play a major role in the dissemination of antibiotic resistance gene cassettes among Gram-negative bacteria. Cassette capture is catalyzed by the integron integrase, whose expression is induced by DNA damage and controlled by the bacterial SOS response in laboratory planktonic cultures. In natural settings, bacteria usually grow in heterogeneous environments known as biofilms, which have very different conditions than planktonic cultures. Integrase regulation has not been investigated in biofilms. Our results showed that in addition to the SOS response, the stringent response (induced upon starvation) is specifically involved in the regulation of class 1 integron integrases in biofilms. This study shows that biofilms are favorable environments for integron-mediated acquisition/exchange of antibiotic resistance genes by bacteria and for the emergence of multidrug-resistant bacteria.

FIG 1

IntI1 excision activity under biofilm conditions. IntI1 excision recombination activity was estimated by determining the frequency of emergence of tobramycin resistance as a result of recombination between the attC sites of the attC_aadA7-cat(T4)-attC_VCR-aac(6′)-Ib gene cassette array carried on plasmid p6851. The intI1 gene was expressed from its own promoter (pZE1-intI1). Excision frequency was estimated under biofilm conditions (B) and in planktonic culture (P) with the wild-type strain MG1656 F′ carrying both p6851 and pZE1-intI1. Assays were done at least 9 times each. The bottom and top of the box indicate the first and third quartile respectively. The median is shown as a horizontal line inside the box, and the maximum and minimum values as the ends of the whisker.

FIG 2

Complementation experiment with relA and lon mutants. The activity level of the derepressed integrase promoter PintI1* was estimated by β-galactosidase assay. (A) Results for complementation of relA. MG1656ΔrelA/spoT F′/pPintI1*-lacZ also carried pZS*tetR11-mcs1 (pZS-mcs1) or pZS*tetR11-relA (pZS-relA). (B) Results for complementation of lon. MG1656Δlon F′/pPintI1*-lacZ also carried pZS*tetR11-mcs1 or pZS*tetR11-lon (pZS-lon); Strains were grown for 24 h under biofilm conditions or planktonic culture in the absence or presence of 0.2 mM anhydrotetracycline (ATc; induction of RelA or Lon protein synthesis). Error bars indicate the standard deviations of the results from 6 different assays.

FIG 3

Effect of the stringent response on intI1 and sfiA expression under biofilm conditions. The activity levels of the PintI1 and PsfiA promoters, expressed as Miller units, were estimated by β-galactosidase assay in 24-h planktonic and biofilm cultures of the wild-type (WT) strain MG1656 F′ and its ΔrelA/spoT and Δlon derivatives, as indicated. The bacteria carried plasmid pPintI1-lacZ (A) or PsfiA-lacZ (B). Error bars indicate the standard deviations of the results from at least 6 different assays.

FIG 4

Proposed mechanism of class 1 integron regulation via stringent response under biofilm conditions. In biofilms, upon nutrient starvation, the alarmone (p)ppGpp, synthesized by the RelA and SpoT proteins, would mediate the inhibition of replication initiation and transcription of specific genes, thereby stalling the RNA polymerase and leading to the generation of single-stranded DNA (ssDNA) and, thus, to mild induction of the SOS response, resulting in autoproteolysis of the LexA dimer bound to the PintI1 promoter and expression of intI1 (a) and the inhibition of the exopolyphosphatase (PPX) activity, resulting in the accumulation of inorganic polyphosphate (poly-P) via the polyphosphate kinase (PPK), whereupon poly-P would bind the Lon protease to form the poly-P–Lon complex that would regulate the degradation of an unknown regulator of derepressed PintI1 (b).