Oriol, C. | Search bioRxiv

bioRxiv2024

High-throughput Tn-seq screens identify both known and novel Pseudomonas putida KT2440 genes involved in metal resistance

Chemical waste with toxic effects is released into the environment by industrial and urban activities. Pseudomonas putida, a rhizosphere bacterium, harbors a wide variety of genes capable of degrading hydrocarbons and xenobiotic compounds in its natural environment. This bacterium harbors also a large set of metal resistance genes. Most studies that identify genes involved in metal resistance in P. putida focus on over/underexpressed genes and may miss other genes important for metal resistance whose expression does not change. In this study, we used a Tn-seq approach to determine the essential genome of P. putida required for growth in the presence of an excess of metals in a culture medium. Tn-seq enables the detection of mutants with reduced or increased fitness in the presence of metal excess. We validated our screen by identifying known metal resistance gene such as czcA-1 (PP_0043), cadA-3 (PP_5139), cadR (PP_5140) and pcoA2 (PP_5380). Their mutants were underrepresented in the presence of zinc, cadmium (for cadA-3 and cadR) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay that PP_5337 and PP_0887 are putative transcriptional regulators involved in copper and cadmium resistance, respectively, in P. putida. The study revealed the role of two genes, PP_1663 and PP_5002, in cadmium and cobalt resistance respectively. This is the first evidence linking these genes to metal resistance and highlights the incomplete understanding of metal resistance mechanisms in P. putida.

microbiology↗

bioRxiv2023

Small RNA regulation of an essential process induces bacterial resistance to aminoglycosides during oxidative stress

Fe-S clusters are essential cofactors involved in many reactions across all domains of life. In Escherichia coli and other enterobacteria, Fe-S cluster synthesis involves two machineries: Isc and Suf. While Isc functions as a housekeeping system, Suf is activated under stress conditions such as iron starvation or oxidative stress. Interestingly, cells functioning under Suf show reduced entry of aminoglycosides, leading to resistance to these antibiotics. The transcriptional regulator IscR, itself an Fe-S cluster containing protein, controls the transition between Isc and Suf machineries. Noteworthy, IscR has a critical impact on the virulence of various bacterial pathogens by regulating both Fe-S biogenesis and other pathways directly linked to host adaptation. Here, we discovered that two small regulatory RNAs (sRNAs), FnrS and OxyS, control iscR expression by base-pairing to the 5 UTR of the iscR mRNA. Remarkably, these sRNAs act in opposite ways and in opposite conditions: FnrS, expressed in anaerobiosis, represses the expression of iscR while OxyS, expressed during oxidative stress, activates it. Using an E. coli strain experiencing protracted oxidative stress, we further demonstrate that iscR expression is rapidly and significantly enhanced in the presence of OxyS. Strikingly, we further show that OxyS induces resistance to aminoglycosides during oxidative stress through this unexpected regulation of Fe-S clusters biogenesis, revealing a new role for this sRNA. Significance StatementThis study sheds light on the regulatory mechanisms controlling the synthesis of essential Fe-S clusters in bacteria, revealing unexpected roles for two small RNAs (FnrS and OxyS) in modulating the expression of the transcriptional regulator IscR. The findings suggest that this regulatory network could lead bacterial resistance to aminoglycoside antibiotics during oxidative stress, a condition associated with chronic infections. Ultimately, this work highlights the importance of understanding the intricate regulatory networks controlling bacterial metabolism and adaptation to stress, which could have significant implications for public health.

microbiology↗