Biology subjects

Da Re, S.

Publications and source records attributed to Da Re, S..

5 recordsLinked to original sources

bioRxiv2025

The bacterial SOS response promotes the expression of the transposase encoded by ISCR mobile genetic elements

Insertion sequences (IS) are widely involved in bacterial genomic plasticity by disrupting, adding, moving genomic sequences, or by activating or extinguishing gene expression. A specific family of IS, ISCR (for insertion sequence of Common Region), is thought to be involved in the dissemination of antibiotic resistance genes (ARG). While some ISCR members are commonly found in bacteria isolated in clinical settings and can contribute to downstream ARG expression, the mechanisms regulating the ISCR-encoded transposases expression have remained uncharacterized. Here, we investigated the expression of the transposase gene of ISCR1, ISCR2 and ISCR8, and its regulation in Escherichia coli. Using in silico analyses and in vitro experiments, we showed that expression levels were extremely low, as observed for most IS transposases. We further demonstrated the direct role of DNA damages and the key SOS response repressor, LexA, in controlling the activity of transposase promoter. These results provide evidence that the mobility of at least some ISCR elements may be promoted upon bacterial exposure to antibiotics inducing the SOS response. IMPORTANCEMobile genetic elements are the most prevalent cause of antibiotic resistance emergence. Among these mobile elements, insertion sequences (IS) are well known to allow the dissemination of antibiotic resistance genes through the action of their tranposase. Here, we studied the regulation of the transposase expression in a specific family of IS, the ISCR family, some members of which are known to be involved in antibiotic resistance. Characterizing the regulation of transposase expression is an important starting point for understanding how these IS can contribute, through their movement, to the spread and expression of antibiotic resistance. This knowledge is necessary if we are to hope to prevent this spread one day.

microbiology↗

bioRxiv2025

Dual regulatory role of IS91-encoded Orf121 in IS91 transposition

Prokaryotic insertion sequences (IS) are pivotal in the propagation of bacterial multidrug resistance, with IS91 notably linked to virulence and antibiotic resistance genes. However, the precise mechanism by which IS91 contributes to gene dissemination remains elusive. Unique among its family, IS91 features a small open reading frame (orf) upstream of the tnpA transposase gene, potentially encoding a 121-amino acid protein, orf121, which may be translationally coupled with tnpA. Using a genetic system based on the mating-out assay in Escherichia coli, we explored the role of orf121 in the in vivo transposition of IS91. Our findings indicate that the overlap between orf121 and tnpA is crucial for tnpA transcription, with both being primarily transcribed as bicistronic mRNAs from the Porf121 promoter. Additionally, the expression of orf121 (whether in cis or trans) significantly reduces the frequency of IS91 transposition and the rate of one-ended transposition. Furthermore, only the single-stranded DNA circles of IS91 intermediates can integrate into new target sequences, and Orf121 negatively influences this insertion step. In summary, orf121 acts as a negative regulator of transposition while ensuring the expression of tnpA, thus providing insights into the complex mechanisms underlying IS91-mediated gene dissemination and its potential role in antibiotic resistance propagation.

molecular biology↗

bioRxiv2023

Hospital and urban wastewaters shape the structure and active resistome of environmental biofilms

BackgroundDemonstration of the transfer, dynamics, and regulation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in a complex environmental matrix is yet experimentally challenging, with many essential open questions such as how and where transfer and dissemination of ARGs happens in nature. The extent and conditions of MGEs transfer that carry ARGs is still largely unexplored in natural environments and microbial communities. Biofilms are structures that include high density multi-species bacterial communities embedded in self-produced extracellular polymeric substances (EPS) constituting a matrix that facilitates gene transfer and where bacteria exhibit high tolerance to stress and to antibiotics. In this study we implemented a sampling and analysis approach that allows phenotypic and genomic analyses of in situ and reconstituted in vitro hospital and urban wastewater (WW) biofilms. To assess the potential of hospital and urban WW biofilms to efficiently disseminate ARGs in the WW system, we explored the EPS within the biofilm matrix and assessed the expression of the resistome (ARGs) and mobilome (MGEs) by metatranscriptomics. ResultsWe first showed that a) the composition of EPS differs depending on their growth environment (in situ and in vitro) and their sampling origin (hospital vs urban WW) and that b) a low amount of ciprofloxacin impacted the composition of the EPS. Next, the metatranscriptomic approach showed that a) expression of ARGs and MGEs increase upon adding a low amount of ciprofloxacin for biofilms from hospital WW but not for those from urban WW and b) that expression of specific plasmids that carry individual or multiple ARGs varies depending on the WW origins of the biofilms. When the same plasmids were expressed in both, urban and hospital WW biofilms, they carried and expressed different ARGs. ConclusionWe show that hospital and urban wastewaters shape the structure and active resistome of environmental biofilms, and we confirmed that hospital WW is an important hot spot for the dissemination and selection of AMR. The different responses to antibiotic pressure in hospital vs urban biofilms, coupled with differences in biofilm structure helps delineate distinct characteristics of hospital and urban WW biofilms highlighting the relationships between the resistome and its expression in environmental biofilms and their surrounding ecosystems.

microbiology↗

bioRxiv2022

Conjugative IncC plasmid entry triggers the SOS response and promotes effective transfer of the integrative antibiotic resistance element SGI1

The broad host range IncC plasmid family and the integrative mobilizable Salmonella Genomic Island 1 (SGI1) and its derivatives enable the spread of medically-important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCEAntimicrobial resistance has become a major public health issue, particularly with the increase in multidrug resistance (MDR) in both animal and human pathogenic bacteria, and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements such as conjugative plasmids and integrative elements conferring multidrug resistance is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically-important resistance genes (e.g., ESBLs, and carbapenemases). A better knowledge of the complex biology of these broad-host range mobile elements will help to understand the dissemination of antimicrobial resistance genes that occurred across{gamma} -proteobacteria borders.

microbiology↗

bioRxiv2021

Microniches in biofilm depth are hot-spots for antibiotic resistance acquisition in response to in situ stress

Class 1 integrons play a major role in antibiotic resistance dissemination among Gram-negative bacteria. They are genetic platforms able to capture, exchange and express antibiotic resistance gene cassettes. The integron integrase, whose expression is regulated by the bacterial SOS response, is the key element of the integron catalyzing insertion/excision/shuffling of gene cassettes. We previously demonstrated that the basal level of integrase expression and in consequence, its activity, is increased via the starvation-induced stringent response in the biofilm population. However, biofilms are heterogeneous environments where bacteria are under various physiological states. Here we thus analyzed at the bacterial level, the SOS response and integrase expression within the biofilm, using confocal microscopy and flow cytometry. We showed that in the absence of exogenous stress, only a small number of bacteria (~ 1%) located in the depth of the biofilm induce the SOS-response leading to a high level of integrase expression, through both a stringent response-dependent and -independent manner. Our results thus indicate that few bacteria located in microniches of the biofilm depth undergo sufficient endogenous stress to promote the acquisition of antibiotic resistance, forming a reservoir of bacteria ready to rapidly resist antibiotic treatments.

microbiology↗