Role of RelA-synthesized (p)ppGpp and ROS-induced mutagenesis in de novo acquisition of antibiotic resistance in E. coli.

The stringent response of bacteria to starvation and stress also fulfills a role in addressing the threat of antibiotics. Within this stringent response, (p)ppGpp, synthesized by RelA or SpoT, functions as a global alarmone. However, the effect of this (p)ppGpp on resistance development is poorly understood. Here, we show that knockout of relA or rpoS curtails resistance development against bactericidal antibiotics. The emergence of mutated genes associated with starvation and (p)ppGpp, among others, indicates the activation of stringent responses. The growth rate is decreased in ΔrelA-resistant strains due to the reduced ability to synthesize (p)ppGpp and the persistence of deacylated tRNA impeding protein synthesis. Sluggish cellular activity causes decreased production of reactive oxygen species (ROS), thereby reducing oxidative damage, leading to weakened DNA mismatch repair, potentially reducing the generation of mutations. These findings offer new targets for mitigating antibiotic resistance development, potentially achieved through inhibiting (p)ppGpp or ROS synthesis.

The Effect of the Stringent Response and Oxidative Stress Response on Fitness Costs of De Novo Acquisition of Antibiotic Resistance.

Resistance evolution during exposure to non-lethal levels of antibiotics is influenced by various stress responses of bacteria which are known to affect growth rate. Here, we aim to disentangle how the interplay between resistance development and associated fitness costs is affected by stress responses. We performed de novo resistance evolution of wild-type strains and single-gene knockout strains in stress response pathways using four different antibiotics. Throughout resistance development, the increase in minimum inhibitory concentration (MIC) is accompanied by a gradual decrease in growth rate, most pronounced in amoxicillin or kanamycin. By measuring biomass yield on glucose and whole-genome sequences at intermediate and final time points, we identified two patterns of how the stress responses affect the correlation between MIC and growth rate. First, single-gene knockout E. coli strains associated with reactive oxygen species (ROS) acquire resistance faster, and mutations related to antibiotic permeability and pumping out occur earlier. This increases the metabolic burden of resistant bacteria. Second, the ΔrelA knockout strain, which has reduced (p)ppGpp synthesis, is restricted in its stringent response, leading to diminished growth rates. The ROS-related mutagenesis and the stringent response increase metabolic burdens during resistance development, causing lower growth rates and higher fitness costs.

Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant.

IMPORTANCE: WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

Reactive oxygen species accelerate de novo acquisition of antibiotic resistance in E. coli.

Reactive oxygen species (ROS) produced as a secondary effect of bactericidal antibiotics are hypothesized to play a role in killing bacteria. If correct, ROS may play a role in development of de novo resistance. Here we report that single-gene knockout strains with reduced ROS scavenging exhibited enhanced ROS accumulation and more rapid acquisition of resistance when exposed to sublethal levels of bactericidal antibiotics. Consistent with this observation, the ROS scavenger thiourea in the medium decelerated resistance development. Thiourea downregulated the transcriptional level of error-prone DNA polymerase and DNA glycosylase MutM, which counters the incorporation and accumulation of 8-hydroxy-2'-deoxyguanosine (8-HOdG) in the genome. The level of 8-HOdG significantly increased following incubation with bactericidal antibiotics but decreased after treatment with the ROS scavenger thiourea. These observations suggest that in E. coli sublethal levels of ROS stimulate de novo development of resistance, providing a mechanistic basis for hormetic responses induced by antibiotics.

The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis.

BACKGROUND: Bacteria can acquire resistance through DNA mutations in response to exposure to sub-lethal concentrations of antibiotics. According to the radical-based theory, reactive oxygen species (ROS), a byproduct of the respiratory pathway, and oxidative stress caused by reactive metabolic byproducts, play a role in cell death as secondary killing mechanism. In this study we address the question whether ROS also affects development of resistance, in the conditions that the cells is not killed by the antibiotic. RESULTS: To investigate whether oxygen and ROS affect de novo acquisition of antibiotic resistance, evolution of resistance due to exposure to non-lethal levels of antimicrobials was compared in E. coli wildtype and ΔoxyR strains under aerobic and anaerobic conditions. Since Lactococcus lactis (L. lactis) does not have an active electron transport chain (ETC) even in the presence of oxygen, and thus forms much less ROS, resistance development in L. lactis was used to distinguish between oxygen and ROS. The resistance acquisition in E. coli wildtype under aerobic and anaerobic conditions did not differ much. However, the aerobically grown ΔoxyR strain gained resistance faster than the wildtype or anaerobic ΔoxyR. Inducing an ETC by adding heme increased the rate at which L. lactis acquired resistance. Whole genome sequencing identified specific mutations involved in the acquisition of resistance. These mutations were specific for each antibiotic. The lexA mutation in ΔoxyR strain under aerobic conditions indicated that the SOS response was involved in resistance acquisition. CONCLUSIONS: The concept of hormesis can explain the beneficial effects of low levels of ROS and reactive metabolic byproducts, while high levels are lethal. DNA repair and mutagenesis may therefore expedite development of resistance. Taken together, the results suggest that oxygen as such barely affects resistance development. Nevertheless, non-lethal levels of ROS stimulate de novo acquisition of antibiotic resistance.

Genome sequences of 10 new carnation mottle virus variants.

Here, we report the genome sequences of 10 Carnation mottle virus variants. Six variants originated from a single proprietary carnation cultivar, and four were derived from four different proprietary cultivars. All variants showed nucleotide differences, but the last four did not show any variation at the amino acid level.

The protein modifier SUMO is critical for integrity of the Arabidopsis shoot apex at warm ambient temperatures.

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis - a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype.

Increased comparability between RNA-Seq and microarray data by utilization of gene sets.

The field of transcriptomics uses and measures mRNA as a proxy of gene expression. There are currently two major platforms in use for quantifying mRNA, microarray and RNA-Seq. Many comparative studies have shown that their results are not always consistent. In this study we aim to find a robust method to increase comparability of both platforms enabling data analysis of merged data from both platforms. We transformed high dimensional transcriptomics data from two different platforms into a lower dimensional, and biologically relevant dataset by calculating enrichment scores based on gene set collections for all samples. We compared the similarity between data from both platforms based on the raw data and on the enrichment scores. We show that the performed data transforms the data in a biologically relevant way and filters out noise which leads to increased platform concordance. We validate the procedure using predictive models built with microarray based enrichment scores to predict subtypes of breast cancer using enrichment scores based on sequenced data. Although microarray and RNA-Seq expression levels might appear different, transforming them into biologically relevant gene set enrichment scores significantly increases their correlation, which is a step forward in data integration of the two platforms. The gene set collections were shown to contain biologically relevant gene sets. More in-depth investigation on the effect of the composition, size, and number of gene sets that are used for the transformation is suggested for future research.

Effects of a previously selected antibiotic resistance on mutations acquired during development of a second resistance in Escherichia coli.

BACKGROUND: The effect of mutations conferring antibiotic resistance can depend on the genetic background. To determine if a previously de novo acquired antibiotic resistance influences the adaptation to a second antibiotic, antibiotic resistance was selected for by exposure to stepwise increasing sublethal levels of amoxicillin, enrofloxacin, kanamycin, or tetracycline. E. coli populations adapted to either a single or two antibiotics sequentially were characterized using whole genome population sequencing and MIC measurements. RESULTS: In a wild-type background, adaptation to any of the antibiotics resulted in the appearance of well-known mutations, as well as a number of mutated genes not known to be associated with antibiotic resistance. Development of a second resistance in a strain with an earlier acquired resistance to a different antibiotic did not always result in the appearance of all mutations associated with resistance in a wild-type background. In general, a more varied set of mutations was acquired during secondary adaptation. The ability of E. coli to maintain the first resistance during this process depended on the combination of antibiotics used. The maintenance of mutations associated with resistance to the first antibiotic did not always predict the residual MIC for that compound. CONCLUSIONS: In general, the data presented here indicate that adaptation to each antibiotic is unique and independent. The mutational trajectories available in already resistant cells appear more varied than in wild-type cells, indicating that the genetic background of E. coli influences resistance development. The observed mutations cannot always fully explain the resistance pattern observed, indicating a crucial role for adaptation on the gene expression level in de novo acquisition of antibiotic resistance.

Extreme slow growth as alternative strategy to survive deep starvation in bacteria.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call 'oligotrophic growth state', provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.