scholarly journals Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
David G. Christensen ◽  
James S. Orr ◽  
Christopher V. Rao ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Bacterial Growth ◽  
Genetic Manipulation ◽  
Cell Number ◽  
Lysine Acetylation ◽  
Protein Acetylation ◽  
Complex Media ◽  
Magnesium Supplementation ◽  
Content Type

ABSTRACT Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth. IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.

scholarly journals Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
David G. Christensen ◽  
Jesse G. Meyer ◽  
Jackson T. Baumgartner ◽  
Alexandria K. D’Souza ◽  
William C. Nelson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Substrate Specificity ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Lysine Acetylation ◽  
Acetyl Phosphate ◽  
Protein Acetylation ◽  
Content Type ◽  
E Coli

ABSTRACT Posttranslational modifications, such as Nε-lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either nonenzymatically or enzymatically. The nonenzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donor to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in Escherichia coli. Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the posttranslational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration. Together, these results emphasize distinct and specific nonenzymatic and enzymatic protein acetylation mechanisms present in bacteria. IMPORTANCE Nε-Lysine acetylation is one of the most abundant and important posttranslational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, Nε-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed an E. coli strain that lacked both known acetylation mechanisms to identify four new Nε-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate nonenzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellum-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial posttranslational modification.

scholarly journals Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Birgit Schilling ◽  
Nathan Basisty ◽  
David G. Christensen ◽  
Dylan Sorensen ◽  
James S. Orr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Regulatory Mechanism ◽  
Growth Conditions ◽  
Lysine Acetylation ◽  
Label Free ◽  
Content Type ◽  
E Coli ◽  
Regulatory Mode ◽  
Specific Sugar

ABSTRACT Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli. IMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

scholarly journals Serine-Threonine Kinases Encoded by SplithipAHomologs Inhibit Tryptophanyl-tRNA Synthetase

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Stine Vang Nielsen ◽  
Kathryn Jane Turnbull ◽  
Mohammad Roghanian ◽  
Rene Bærentsen ◽  
Maja Semanjski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Gene Family ◽  
Sequence Similarity ◽  
Trna Synthetase ◽  
Bacterial Toxin ◽  
Content Type ◽  
Multidrug Tolerance ◽  
K 12

ABSTRACTType II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact.hipBAofEscherichia colistrain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction ofhipAinhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed thattrpSin high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT ofE. coliO127 is a kinase that phosphorylates tryptophanyl-tRNA synthetasein vitroat a conserved serine residue. Consistently, induction ofhipTinhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream fromhipT, calledhipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipSin vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additionalhipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus,hipBSTconstitutes a novel family of tricomponent TA modules wherehipAhas been split into two genes,hipSandhipT, that function as a novel type of TA pair.IMPORTANCEBacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified washipAofEscherichia colistrain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. ThehipAgene encodes the toxin of thehipBATA module, whilehipBencodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family,hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

scholarly journals Phenotypic Detection of Plasmid-Mediated Colistin Resistance in Enterobacteriaceae

2019 ◽  
Vol 58 (3) ◽  
Author(s):  
Edgar Gonzales Escalante ◽  
Katherine Yauri Condor ◽  
Jose A. Di Conza ◽  
Gabriel O. Gutkind
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Serratia Marcescens ◽  
Bacterial Growth ◽  
Screening Test ◽  
Colistin Resistance ◽  
Content Type ◽  
E Coli

ABSTRACT The aim of this work was to evaluate an easy-to-perform assay based upon inhibition of mobile colistin resistance (MCR) activity by EDTA. We included 92 nonrelated isolates of Enterobacteriaceae (74 Escherichia coli, 17 Klebsiella pneumoniae, and 1 Serratia marcescens). Our proposed method is based on a modification of the colistin agar-spot screening test (CAST), a plate containing 3 μg/ml colistin, by adding an extra plate of colistin agar-spot supplemented with EDTA (eCAST). Bacterial growth was evaluated after 24 h of incubation at 35°C. All the colistin-resistant isolates showed development on the CAST plates. Colistin-resistant K. pneumoniae without mcr-1 and S. marcescens also grew on the eCAST plates. In contrast, colistin-resistant MCR-producing E. coli was not able to grow in eCAST plates. The combined CAST/eCAST test could provide a simple and easy-to-perform method to differentiate MCR-producing Enterobacteriaceae from those in which colistin resistance is mediated by chromosomal mechanisms.

scholarly journals Extension of Pharmacokinetic/Pharmacodynamic Time-Kill Studies To Include Lipopolysaccharide/Endotoxin Release from Escherichia coli Exposed to Cefuroxime

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Anders Thorsted ◽  
Eva Tano ◽  
Kia Kaivonen ◽  
Jan Sjölin ◽  
Lena E. Friberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Mean Transit Time ◽  
Integrated Analysis ◽  
Antibiotic Exposure ◽  
Bacterial Killing ◽  
Content Type ◽  
Time Courses ◽  
Time Kill

ABSTRACT The release of inflammatory bacterial products, such as lipopolysaccharide (LPS)/endotoxin, may be increased upon the administration of antibiotics. An improved quantitative understanding of endotoxin release and its relation to antibiotic exposure and bacterial growth/killing may be gained by an integrated analysis of these processes. The aim of this work was to establish a mathematical model that relates Escherichia coli growth/killing dynamics at various cefuroxime concentrations to endotoxin release in vitro. Fifty-two time-kill experiments informed bacterial and endotoxin time courses and included both static (0×, 0.5×, 1×, 2×, 10×, and 50× MIC) and dynamic (0×, 15×, and 30× MIC) cefuroxime concentrations. A model for the antibiotic-bacterium interaction was established, and antibiotic-induced bacterial killing followed a sigmoidal Emax relation to the cefuroxime concentration (MIC-specific 50% effective concentration [EC50], maximum antibiotic-induced killing rate [Emax] = 3.26 h−1 and γ = 3.37). Endotoxin release was assessed in relation to the bacterial processes of growth, antibiotic-induced bacterial killing, and natural bacterial death and found to be quantitatively related to bacterial growth (0.000292 endotoxin units [EU]/CFU) and antibiotic-induced bacterial killing (0.00636 EU/CFU). Increased release following the administration of a second cefuroxime dose was described by the formation and subsequent antibiotic-induced killing of filaments (0.295 EU/CFU). Release due to growth was instantaneous, while release due to antibiotic-induced killing was delayed (mean transit time of 7.63 h). To conclude, the in vitro release of endotoxin is related to bacterial growth and antibiotic-induced killing, with higher rates of release upon the killing of formed filaments. Endotoxin release over 24 h is lowest when antibiotic exposure rapidly eradicates bacteria, while increased release is predicted to occur when growth and antibiotic-induced killing occur simultaneously.

scholarly journals Rapid Detection of Fosfomycin Resistance in Escherichia coli

2018 ◽  
Vol 57 (1) ◽  
Author(s):  
Patrice Nordmann ◽  
Laurent Poirel ◽  
Linda Mueller
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Test Performance ◽  
Color Change ◽  
Ph Indicator ◽  
Content Type ◽  
E Coli ◽  
Yellow Color ◽  
Fosfomycin Resistance ◽  
User Friendly

ABSTRACT The rapid fosfomycin/Escherichia coli NP test was developed to detect fosfomycin resistance in E. coli isolates. The test is based on glucose metabolization and the detection of bacterial growth in the presence of fosfomycin at 40 µg/ml. Bacterial growth is visually detectable by an orange-to-yellow color change of red phenol, a pH indicator. A total of 100 E. coli isolates, among which 22 were fosfomycin resistant, were used to evaluate the test performance. The sensitivity and specificity of the test were 100% and 98.7%, respectively. This new test is user friendly, sensitive and specific, and its results are obtained in 1 h 30 min.

scholarly journals Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Anne Kijewski ◽  
Ingun Lund Witsø ◽  
Hildegunn Iversen ◽  
Helene Thorsen Rønning ◽  
Trine L'Abée-Lund ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vitamin K ◽  
Bacterial Growth ◽  
Shiga Toxin ◽  
Treatment Options ◽  
Inhibitory Effect ◽  
Enterohemorrhagic Escherichia Coli ◽  
Lytic Cycle ◽  
Content Type ◽  
Negative Effect

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest. IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

scholarly journals Behavior of Escherichia coli in a Heterogeneous Gelatin-Dextran Mixture

2013 ◽  
Vol 79 (9) ◽  
pp. 3126-3128 ◽  
Author(s):  
K. Boons ◽  
L. Mertens ◽  
E. Van Derlinden ◽  
C. C. David ◽  
J. Hofkens ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Confocal Laser Scanning Microscopy ◽  
Bacterial Growth ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Absolute Concentration ◽  
Content Type

ABSTRACTIn a gelatin-dextran mixture, changing the (relative and/or absolute) concentration of the components leads to the formation of different microstructures. Confocal laser scanning microscopy illustrated that the nature of the microstructure determines the location and morphology ofEscherichia colicolonies. Observations indicate that bacterial growth preferentially occurs in the dextran phase, regardless of the microstructure.

scholarly journals Global Analysis of the Specificities and Targets of Endoribonucleases from Escherichia coli Toxin-Antitoxin Systems

mBio ◽  
2021 ◽  
Author(s):  
Peter H. Culviner ◽  
Isabel Nocedal ◽  
Sarah M. Fortune ◽  
Michael T. Laub
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Global Analysis ◽  
Content Type ◽  
Almost All

Toxin-antitoxin (TA) systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacterized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets.

scholarly journals Crucial Role of ppGpp in the Resilience of Escherichia coli to Growth Disruption

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e01132-20
Author(s):  
Clément Patacq ◽  
Nicolas Chaudet ◽  
Fabien Létisse
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Crucial Role ◽  
Stringent Response ◽  
Content Type ◽  
E Coli ◽  
Changing Environments ◽  
Growth Recovery ◽  
Growth Disruption

ABSTRACTBacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. The results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and, more surprisingly, a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth, and the main effect involved in the return to the basal level was identified by flux calculation as growth dilution. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology.IMPORTANCE The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependence on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.

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