scholarly journals Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Prashant Karki ◽  
Sayed Golam Mohiuddin ◽  
Pouria Kavousi ◽  
Mehmet A. Orman
Keyword(s):  
Stress Factors ◽  
General Notion ◽  
Late Stationary Phase ◽  
Complete Understanding ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Phenotype Microarrays ◽  
Ph Conditions ◽  
Slow Growing

ABSTRACT Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

scholarly journals Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

2019 ◽  
Author(s):  
Prashant Karki ◽  
Mehmet A. Orman
Keyword(s):  
Therapeutic Strategies ◽  
Stress Factors ◽  
General Notion ◽  
Late Stationary Phase ◽  
Complete Understanding ◽  
Persister Cells ◽  
E Coli ◽  
Phenotype Microarrays ◽  
Ph Conditions ◽  
Slow Growing

ABSTRACTBacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance towards antibiotics. Persisters have been linked to the recalcitrance of biofilm related infections; hence, a complete understanding of the physiology of persisters can lead to improvement of therapeutic strategies associated with such infections. Mechanisms pertaining to persister formation are known to be related to stress response pathways triggered from intra- or extra-cellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH- induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, here we studied the effects of various osmolytes and pH conditions onEscherichia colipersistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite and acidic pH, significantly reduced persister formation inE. colicompared to no-osmolyte/no-buffer controls, this reduction in persister levels was lessened in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

scholarly journals Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to β-Lactams

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Pavel Kudrin ◽  
Vallo Varik ◽  
Sofia Raquel Alves Oliveira ◽  
Jelena Beljantseva ◽  
Teresa Del Peso Santos ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Growth Stress ◽  
Amino Acid Starvation ◽  
Bacterial Metabolism ◽  
Persister Cells ◽  
Biochemical System ◽  
Content Type ◽  
E Coli ◽  
Bacterial Cultures ◽  
A Site

ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia coli(p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purifiedE. colicomponents, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

scholarly journals Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

2014 ◽  
Vol 58 (4) ◽  
pp. 2089-2097 ◽  
Author(s):  
Lee W. Goneau ◽  
Nigel S. Yeoh ◽  
Kyle W. MacDonald ◽  
Peter A. Cadieux ◽  
Jeremy P. Burton ◽  
...  
Keyword(s):  
Differential Susceptibility ◽  
Type I ◽  
Antibiotic Tolerance ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Multidrug Tolerance ◽  
External Stimuli ◽  
Susceptibility Patterns

ABSTRACTPersister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed betweenStaphylococcus saprophyticusandEscherichia coliuropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancyper secannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenicE. coliisolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

scholarly journals Sinorhizobium meliloti, a Slow-Growing Bacterium, Exhibits Growth Rate Dependence of Cell Size under Nutrient Limitation

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Xiongfeng Dai ◽  
Zichu Shen ◽  
Yiheng Wang ◽  
Manlu Zhu
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Cell Size ◽  
Sinorhizobium Meliloti ◽  
Cell Cycle Progression ◽  
Bacterial Species ◽  
Progression Rate ◽  
Content Type ◽  
E Coli ◽  
Slow Growing

ABSTRACTBacterial cells need to coordinate the cell cycle with biomass growth to maintain cell size homeostasis. For fast-growing bacterial species likeEscherichia coliandBacillus subtilis, it is well-known that cell size exhibits a strong dependence on the growth rate under different nutrient conditions (known as the nutrient growth law). However, cell size changes little with slow growth (doubling time of >90 min) forE. coli, posing the interesting question of whether slow-growing bacteria species also observe the nutrient growth law. Here, we quantitatively characterize the cell size and cell cycle parameter of a slow-growing bacterium,Sinorhizobium meliloti, at different nutrient conditions. We find thatS. melilotiexhibits a threefold change in its cell size when its doubling time varies from 2 h to 6 h. Moreover, the progression rate of its cell cycle is much longer than that ofE. coli, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate. Our study shows that the nutrient growth law holds robustly regardless of the growth capacity of the bacterial species, generalizing its applicability among the bacterial kingdom.IMPORTANCEThe dependence of cell size on growth rate is a fundamental principle in the field of bacterial cell size regulation. Previous studies of cell size regulation mainly focus on fast-growing bacterial species such asEscherichia coliandBacillussubtilis. We find here thatSinorhizobium meliloti, a slow-growing bacterium, exhibits a remarkable growth rate-dependent cell size pattern under nutrient limitation, generalizing the applicability of the empirical nutrient growth law of cell size. Moreover,S. melilotiexhibits a much slower speed of cell cycle progression thanE. colidoes, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate.

scholarly journals Zinc Acetate Potentiates the Action of Tosufloxacin against Escherichia coli Biofilm Persisters

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Masaru Usui ◽  
Hayato Yokoo ◽  
Yutaka Tamura ◽  
Chie Nakajima ◽  
Yasuhiko Suzuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Zinc Acetate ◽  
Bacterial Infections ◽  
Sos Response ◽  
Persister Cells ◽  
Major Health ◽  
Content Type ◽  
E Coli ◽  
Potential Strategy ◽  
Multiple Antibiotics

ABSTRACT Formation of bacterial biofilms is a major health threat due to their high levels of tolerance to multiple antibiotics and the presence of persisters responsible for infection relapses. We previously showed that a combination of starvation and induction of SOS response in biofilm led to increased levels of persisters and biofilm tolerance to fluoroquinolones. In this study, we hypothesized that inhibition of the SOS response may be an effective strategy to target biofilms and fluoroquinolone persister cells. We tested the survival of Escherichia coli biofilms to different classes of antibiotics in starved and nonstarved conditions and in the presence of zinc acetate, a SOS response inhibitor. We showed that zinc acetate potentiates, albeit moderately, the activity of fluoroquinolones against E. coli persisters in starved biofilms. The efficacy of zinc acetate to increase fluoroquinolone activity, particularly that of tosufloxacin, suggests that such a combination may be a potential strategy for treating biofilm-related bacterial infections.

scholarly journals Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

2016 ◽  
Vol 82 (20) ◽  
pp. 6167-6173 ◽  
Author(s):  
Meenu Katoch ◽  
Rabia Mazmouz ◽  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Russell Pickford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Uv Radiation ◽  
Active Ingredient ◽  
Stress Factors ◽  
Heterologous Production ◽  
Content Type ◽  
E Coli

ABSTRACTMycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacteriumCylindrospermum stagnalePCC 7417 revealed a new gene cluster with homology to MAA synthase fromNostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylAtomylE), compared to the four found in other MAA gene clusters. Heterologous expression ofmylAtomylEinEscherichia coliresulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced inE. coliand structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods.IMPORTANCEMycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs inE. coliis also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

scholarly journals Recombinant Reporter Assay Using Transcriptional Machinery of Mycobacterium tuberculosis

2014 ◽  
Vol 197 (3) ◽  
pp. 646-653 ◽  
Author(s):  
Rajdeep Banerjee ◽  
Paulami Rudra ◽  
Abinit Saha ◽  
Jayanta Mukhopadhyay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Receptor Protein ◽  
Reporter Assay ◽  
Content Type ◽  
E Coli ◽  
Gene Reporter Assay ◽  
Σ Factor ◽  
Slow Growing

Development of anin vivogene reporter assay to assess interactions among the components of the transcription machinery inMycobacterium tuberculosisremains a challenge to scientists due to the tediousness of generation of mutant strains of the extremely slow-growing bacterium. We have developed a recombinant mCherry reporter assay that enables us to monitor the interactions ofMycobacterium tuberculosistranscriptional regulators with its promotersin vivoinEscherichia coli. The assay involves a three-plasmid expression system inE. coliwherein two plasmids are responsible forM. tuberculosisRNA polymerase (RNAP) production and the third plasmid harbors the mCherry reporter gene expression cassette under the control of either a σ factor or a transcriptional regulator-dependent promoter. We observed that the endogenousE. coliRNAP and σ factor do not interfere with the assay. By using the reporter assay, we found that the functional interaction ofM. tuberculosiscyclic AMP receptor protein (CRP) occurs with its own RNA polymerase, not with theE. colipolymerase. Performing the recombinant reporter assay inE. coliis much faster than if performed inM. tuberculosisand avoids the hazard of handling the pathogenic bacterium. The approach could be expanded to develop reporter assays for other pathogenic and slow-growing bacterial systems.

scholarly journals Nitrogen Starvation Induces Persister Cell Formation inEscherichia coli

2018 ◽  
Vol 201 (3) ◽  
Author(s):  
Daniel R. Brown
Keyword(s):  
Stress Responses ◽  
Nitrogen Starvation ◽  
Cell Formation ◽  
Nitrogen Regulation ◽  
Adaptive Responses ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Secondary Messenger ◽  
Regulatory Cascade

ABSTRACTTo cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such asEscherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of therelAgene, the major (p)ppGpp synthetase inE. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starvedE. colisynthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCEBacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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