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Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine., Bock et al. characterise the adaptation of Pseudomonas aeruginosa to the antiseptic octenidine, using whole genome sequencing, gene expression studies and metabolomics. They attribute this increased tolerance to synergistic changes in efflux and plasma membrane composition via mutations in SmvR, the regulator of MFS efflux pump SmvA, and in phospholipid pathway proteins PssA and PgsA.

Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques. Electronic supplementary material The online version of this article (10.1186/s12864-019-5754-6) contains supplementary material, which is available to authorized users.

18S ribosomal RNA methylation by METL-5 regulates lipid oxidation and stress resistance in C. elegans., During stress, global translation is reduced, but specific transcripts are actively translated. How stress-responsive mRNAs are selectively translated is unknown. We show that METL-5 methylates adenosine 1717 on 18S ribosomal RNA in C. elegans, enhancing selective ribosomal binding and translation of specific mRNAs. One of these mRNAs, CYP-29A3, oxidizes the omega-3 polyunsaturated fatty acid eicosapentaenoic acid to eicosanoids, key stress signaling molecules. While metl-5–deficient animals grow normally under homeostatic conditions, they are resistant to a variety of stresses. metl-5 mutant worms also show reduced bioactive lipid eicosanoids and dietary supplementation of eicosanoid products of CYP-29A3 restores stress sensitivity of metl-5 mutant worms. Thus, methylation of a specific residue of 18S rRNA by METL-5 selectively enhances translation of cyp-29A3 to increase production of eicosanoids, and blocking this pathway increases stress resistance. This study suggests that ribosome methylation can facilitate selective translation, providing another layer of regulation of the stress response.

UHPLC-ms/ms quantification of 6mA in gnotobiotic mouse tissues. UHPLC-ms/ms quantification of 6mA in 10 tissues from gnotobiotic mice demonstrates equivalent levels of 6mA. Each bar represents the mean +/− standard error of the mean for 1–3 independent samples (PDF 41 kb)

Quantification of prokaryotic DNA in eukaryotic samples. a) Percentage of bacterial DNA as assessed by real-time RT PCR with 16S rRNA specific primers [54]. Zoomed in plot displayed on the right. Most species tested had less than 1% bacterial contamination. No significant correlation was detected when comparing DNA methylation to bacterial contamination in different eukaryotic species (R2 = 0.08859, p = 0.32). b) Percentage of zebrafish, bacterial, and unknown DNA reads from input and 6mA IP sequencing experiments previously performed [14]. Some bacterial DNA was detected in all samples and it was enriched after 6mA IP but no developmental trend in bacterial concentrations was detected by sequencing analysis. (PDF 31 kb)

Representative UHPLC-ms/ms chromatograms demonstrate 6mA signal and mock correction. Representative UHPLC-ms/ms chromatograms displays the 6mA peak in C. elegans samples (top panel) and mock digestions (lower panel) when signal was a) detected or b) not detected. Mock digestions are subtracted from sample digestions to calculate percent 6mA. c) 6mA concentrations (pmol) in all replicate samples for Fig. 2d, including the mock digestion reactions (black bars) input DNAs (blue bars) and histone H3 IP’d DNAs (red bars). (PDF 57 kb)

Representative UHPLC-ms/ms chromatograms demonstrate 6mA signal and mock correction. Representative UHPLC-ms/ms chromatograms displays the 6mA peak in C. elegans samples (top panel) and mock digestions (lower panel) when signal was a) detected or b) not detected. Mock digestions are subtracted from sample digestions to calculate percent 6mA. c) 6mA concentrations (pmol) in all replicate samples for Fig. 2d, including the mock digestion reactions (black bars) input DNAs (blue bars) and histone H3 IP’d DNAs (red bars). (PDF 57 kb)