Loss of Gre factors leads to phenotypic heterogeneity and cheating in Escherichia coli populations under nitric oxide stress.

Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in Escherichia coli, GreA and GreB, significantly compromised ·NO detoxification due to ·NO-induced phenotypic heterogeneity in ΔgreAΔgreB populations, which did not occur in wild-type cultures. Under normal culturing conditions, both wild-type and ΔgreAΔgreB synthesized transcripts uniformly, whereas treatment with ·NO led to bimodal transcript levels in ΔgreAΔgreB that were unimodal in wild-type. Interestingly, exposure to another toxic metabolite of phagosomes, hydrogen peroxide (H2O2), produced analogous results. Furthermore, we showed that loss of Gre factors led to cheating under ·NO stress where transcriptionally deficient cells benefited from the detoxification activities of the transcriptionally proficient subpopulation. Collectively, these results show that loss of Gre factor activities produces phenotypic heterogeneity under ·NO and H2O2 stress that can yield cheating between subpopulations.IMPORTANCEToxic metabolite stress occurs in a broad range of contexts that are important to human health, microbial ecology, and biotechnology, whereas Gre factors are highly conserved throughout the bacterial kingdom. Here we discovered that loss of Gre factors in E. coli leads to phenotypic heterogeneity under ·NO and H2O2 stress, which we further show with ·NO results in cheating between subpopulations. Collectively, these data suggest that Gre factors play a role in coping with toxic metabolite stress, and that loss of Gre factors can produce cheating between neighbors.

Amino acids can deplete ATP and impair nitric oxide detoxification by Escherichia coli.

Nitric oxide (·NO) is a prevalent antimicrobial that is known to damage iron-containing enzymes in amino acid (AA) biosynthesis pathways. With Escherichia coli, ·NO is detoxified in aerobic environments by Hmp, which is an enzyme that is synthesized de novo in response to ·NO. With this knowledgebase, it is expected that the availability of AAs in the extracellular environment would enhance ·NO detoxification, because AAs would foster translation of Hmp. However, we observed that ·NO detoxification by E. coli was far slower in populations grown and treated in the presence of AAs (AA+) in comparison to those grown and stressed in the absence of AAs (AA-). Further experiments revealed that AA+ populations had difficulty translating proteins under ·NO stress, and that ·NO activated the stringent response in AA+ populations. Additional work revealed significant ATP depletion in ·NO-stressed AA+ cultures that far exceeded that of ·NO-stressed AA- populations. Transcription, translation, and RelA were not found to be significant contributors to the ATP depletion observed, whereas AA import was implicated as a significant ATP consumption pathway. Alleviating ATP depletion while maintaining access to AAs partially restored ·NO detoxification, which suggested that ATP depletion contributed to the translational difficulties observed in ·NO-stressed AA+ populations. These data reveal an unexpected interaction within the ·NO response network of E. coli that stimulates a stringent response by RelA in conditions where AAs are plentiful.

Causes of polymyxin treatment failure and new derivatives to fill the gap.

Polymyxins are a class of antibiotics that were discovered in 1947 from programs searching for compounds effective in the treatment of Gram-negative infections. Produced by the Gram-positive bacterium Paenibacillus polymyxa and composed of a cyclic peptide chain with a peptide-fatty acyl tail, polymyxins exert bactericidal effects through membrane disruption. Currently, polymyxin B and colistin (polymyxin E) have been developed for clinical use, where they are reserved as "last-line" therapies for multidrug-resistant (MDR) infections. Unfortunately, the incidences of strains resistant to polymyxins have been increasing globally, and polymyxin heteroresistance has been gaining appreciation as an important clinical challenge. These phenomena, along with bacterial tolerance to this antibiotic class, constitute important contributors to polymyxin treatment failure. Here, we review polymyxins and their mechanism of action, summarize the current understanding of how polymyxin treatment fails, and discuss how the next generation of polymyxins holds promise to invigorate this antibiotic class.

Translational Fusion to Hmp Improves Heterologous Protein Expression.

Flavohemoglobins, which are widely distributed in prokaryotes and eukaryotes, play key roles in oxygen (O2) transport and nitric oxide (·NO) defense. Hmp is the flavohemoglobin of Escherichia coli, and here we report that the translational fusion of Hmp to the N-terminus of heterologous proteins increases their expression in E. coli. The effect required the fusion of the proteins, and was independent of both the O2-binding and catalytic activity of Hmp. Increased expression was at the translational level, likely to be downstream of initiation, and we observed that as little as the first 100 amino acids of Hmp were sufficient to boost protein production. These data demonstrate the potential of Hmp as an N-terminal fusion tag to increase protein yield, and suggest that the utility of bacterial hemoglobins to biotechnology goes beyond their O2 transport and ·NO detoxification capabilities.

Robustness of nitric oxide detoxification to nitrogen starvation in Escherichia coli requires RelA.

Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (•NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies •NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair •NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to •NO. However, we found that E. coli detoxifies •NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in •NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that •NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during •NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of •NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify •NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of •NO.

Rifamycin antibiotics and the mechanisms of their failure.

Rifamycins are a class of antibiotics that were first discovered in 1957 and are known for their use in treating tuberculosis (TB). Rifamycins exhibit bactericidal activity against many Gram-positive and Gram-negative bacteria by inhibiting RNA polymerase (RNAP); however, resistance is prevalent and the mechanisms range from primary target modification and antibiotic inactivation to cytoplasmic exclusion. Further, phenotypic resistance, in which only a subpopulation of bacteria grow in concentrations exceeding their minimum inhibitory concentration, and tolerance, which is characterized by reduced rates of bacterial cell death, have been identified as additional causes of rifamycin failure. Here we summarize current understanding and recent developments regarding this critical antibiotic class.

Quantifying Nitric Oxide Flux Distributions.

Nitric oxide (NO) is a radical that is used as an attack molecule by immune cells. NO can interact and damage a range of biomolecules, and the biological outcome for bacteria assaulted with NO will be governed by how the radical distributes within their biochemical reaction networks. Measurement of those NO fluxes is complicated by the low abundance and transience of many of its reaction products. To overcome this challenge, we use computational modeling to translate measurements of several biochemical species (e.g., NO, O2, NO2-) into NO flux distributions. In this chapter, we provide a detailed protocol, which includes experimental measurements and computational modeling, to estimate the NO flux distribution in an Escherichia coli culture. Those fluxes will have uncertainty associated with them and we also discuss how further experiments and modeling can be employed for flux refinement.