Unknown,Transcriptomics,Genomics,Proteomics

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Persistence of BW25113 mqsR producing MqsR 2-1 vs. producing wild-type MqsR with ampicillin

ABSTRACT: Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Previously we discerned that MqsR and MqsA of Escherichia coli are a toxin/antitoxin pair that influence persister cell production via their regulation of Hha, CspD, and HokA. Here, to gain more insights into the origin of persisters, we used protein engineering to increase the toxicity of toxin MqsR by reasoning it would be easier to understand the effect of this toxin if it were more toxic. We found that two mutations (K3N and N31Y) increase the toxicity four fold and increase persistence 73 fold compared to native MqsR by making the protein less labile. A whole transcriptome study revealed that the MqsR variant represses acid resistance genes (gadABCEWX and hdeABD), multidrug resistance genes (mdtEF), and osmotic resistance genes (osmEY). Corroborating these microarray results, deletion of rpoS as well as the genes that the master stress response regulator RpoS controls, gadB, gadX, mdtF, and osmY, increased persister formation dramatically to the extent that nearly the whole population became persistent. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems. For the whole-transcriptome study of BW25113 mqsR/pBS(Kan)-mqsR 2-1 versus BW25113 mqsR/pBS(Kan)-mqsR, planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37 M-BM-0C, adjusted the turbidity to 1, and exposed to 20 M-NM-

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Thomas Wood

PROVIDER: E-GEOD-31054 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated, as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB, as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. Overnight cultures of cobB/pCA24N-cobB and cobB/pCA24N were cultured to a turbidity of 0.05 at 600 nm and grown 2 h. Then, 0.1 mM IPTG was added for another 4 h to induce cobB expression, and then the cells were exposed to 20 mM H2O2 for 10 min. Cell pellets were collected and resuspended in RNAlater (Ambion Inc., Austin, TX), and total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). The E. coli GeneChip Genome 2.0 array (Affymetrix, P/N 900551) was used, and cDNA synthesis, fragmentation, and hybridizations were performed as described previously. If the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%) and if their fold change is higher than the standard deviation for the whole genome.

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Persistence of BW25113 mqsR producing MqsR 2-1 vs. producing wild-type MqsR with ampicillin

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