Contribution to Carbapenem Resistance and Fitness Cost of DcuS/DcuR, RcsC/RcsB, and YehU/YehT Two-Component Systems in CTX-M-15-Producing Escherichia coli.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum ß-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

Safety assessment of astaxanthin derived from engineered Escherichia coli K-12 using a 13-week repeated dose oral toxicity study and a prenatal developmental toxicity study in rats.

Astaxanthin is a natural carotenoid with strong antioxidant activity that has been used for decades as a nutrient/color additive and it has recently been marketed as a health supplement. Astaxanthin can be synthesized in a wide range of microalgae, yeast, and bacteria. As genes directing astaxanthin biosynthesis in various organisms have been cloned, this study assessed the safety of astaxanthin crystal produced by Escherichia coli K-12 harboring plasmids carrying astaxanthin biosynthetic genes. The astaxanthin crystal contains a total carotenoid content of 950 mg/g and an astaxanthin content of 795 mg/g. Subchronic oral toxicity and prenatal developmental toxicity of the astaxanthin in rats were conducted in accordance with the Guidelines of Health Food Safety Assessment promulgated by Food and Drug Administration of Taiwan which is based on OECD guidelines 408 and 414. Both male and female Sprague-Dawley (SD) rats (12 for each gender) receiving the astaxanthin crystal at 1.2, 240.0, or 750.0 mg/kg/day in olive oil via oral gavage for 90 days showed no changes in body weight gains, hematology and serum chemistry values and hepatic enzyme stability, organ integrity and organ weight. Except the higher food consumption observed in rats receiving 750.0 mg/g astaxanthin crystal, administration of the astaxanthin crystal to 25-27 pregnant female rats in each group throughout the period of organogenesis (G6-G15) produced no adverse effects on fetal organogenesis. Based on the results, we propose that the no-observable-adverse-effect level (NOAEL) of the astaxanthin crystal extracted from genetically modified E. coli K-12 is 750.0 mg/kg bw/day.

A Comparison of the Costs and Benefits of Bacterial Gene Expression.

To study how a bacterium allocates its resources, we compared the costs and benefits of most (86%) of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. The cost or investment in each protein was estimated from ribosomal profiling data, and the benefit of each protein was measured by assaying a library of transposon mutants. We found that proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host (with a benefit of less than 5% per generation) tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Although some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that at least 13% of the cell's protein is "on standby" in case conditions change.

Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.

Bacterial siderophores are a group of chemically diverse, virulence-associated secondary metabolites whose expression exerts metabolic costs. A combined bacterial genetic and metabolomic approach revealed differential metabolomic impacts associated with biosynthesis of different siderophore structural families. Despite myriad genetic differences, the metabolome of a cheater mutant lacking a single set of siderophore biosynthetic genes more closely approximate that of a non-pathogenic K12 strain than its isogenic, uropathogen parent strain. Siderophore types associated with greater metabolomic perturbations are less common among human isolates, suggesting that metabolic costs influence success in a human population. Although different siderophores share a common iron acquisition function, our analysis shows how a metabolomic approach can distinguish their relative metabolic impacts in E. coli.

Evaluation of handling and reuse approaches for the waste generated from MEA-based CO2 capture with the consideration of regulations in the UAE.

A waste slip-stream is generated from the reclaiming process of monoethanolamine (MEA) based Post-Combustion Capture (PCC). It mainly consists of MEA itself, ammonium, heat-stable salts (HSS), carbamate polymers, and water. In this study, the waste quantity and nature are characterized for Fluor's Econamine FGSM coal-fired CO2 capture base case. Waste management options, including reuse, recycling, treatment, and disposal, are investigated due to the need for a more environmentally sound handling. Regulations, economic potential, and associated costs are also evaluated. The technical, economic, and regulation assessment suggests waste reuse for NOx scrubbing. Moreover, a high thermal condition is deemed as an effective technique for waste destruction, leading to considerations of waste recycling into a coal burner or incineration. As a means of treatment, three secondary-biological processes covering Complete-Mix Activated Sludge (CMAS), oxidation ditch, and trickling filter are designed to meet the wastewater standards in the United Arab Emirates (UAE). From the economic point of view, the value of waste as a NOx scrubbing agent is 6,561,600-7,348,992 USD/year. The secondary-biological treatment cost is 0.017-0.02 USD/ton of CO2, while the cost of an on-site incinerator is 0.031 USD/ton of CO2 captured. In conclusion, secondary biological treatment is found to be the most economical option.

Deep coverage of the Escherichia coli proteome enables the assessment of false discovery rates in simple proteogenomic experiments.

Recent advances in mass spectrometry (MS) have led to increased applications of shotgun proteomics to the refinement of genome annotation. The typical "proteo-genomic" workflows rely on the mapping of peptide MS/MS spectra onto databases derived via six-frame translation of the genome sequence. These databases contain a large proportion of spurious protein sequences which make the statistical confidence of the resulting peptide spectrum matches difficult to assess. Here we performed a comprehensive analysis of the Escherichia coli proteome using LTQ-Orbitrap MS and mapped the corresponding MS/MS spectra onto a six-frame translation of the E. coli genome. We hypothesized that the protein-coding part of the E. coli genome approaches complete annotation and that the majority of six frame-specific (novel) peptide spectrum matches can be considered as false positive identifications. We confirm our hypothesis by showing that the posterior error probability distribution of novel hits is almost identical to that of reversed (decoy) hits; this enables us to estimate the sensitivity, specificity, accuracy, and false discovery rate in a typical bacterial proteo-genomic dataset. We use two complementary computational frameworks for processing and statistical assessment of MS/MS data: MaxQuant and Trans-Proteomic Pipeline. We show that MaxQuant achieves a more sensitive six-frame database search with an acceptable false discovery rate and is therefore well suited for global genome reannotation applications, whereas the Trans-Proteomic Pipeline achieves higher specificity and is well suited for high-confidence validation. The use of a small and well-annotated bacterial genome enables us to address genome coverage achieved in state-of-the-art bacterial proteomics: identified peptide sequences mapped to all expressed E. coli proteins but covered 31.7% of the protein-coding genome sequence. Our results show that false discovery rates can be substantially underestimated even in "simple" proteo-genomic experiments obtained by means of high-accuracy MS and point to the necessity of further improvements concerning the coverage of peptide sequences by MS-based methods.

Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli.

The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

Towards improved assessment of functional similarity in large-scale screens: a study on indel length.

Although insertions and deletions are a common type of evolutionary sequence variation, their origins and their functional consequences have not been comprehensively understood. Most alignment algorithms/programs only roughly reflect the evolutionary processes that result in gaps--which typically require further evaluation. Interestingly, it is widely believed that gaps are the predominant form of sequence variation resulting in structural and functional changes. Thus it is desirable to distinguish between gaps that reflect true point mutations and alignment artifacts when it comes to assessing the functional similarity of proteins based on computational alignments. Here we introduce pair hidden Markov model-based solutions to rapidly assess the statistical significance of gaps in alignments resulting from classical Needleman-Wunsch-like alignment procedures which implement affine gap penalty scoring schemes. Surprisingly, although it has a natural formulation, the emanating Markov chain problem had no known efficient solution thus far. In this article, we present the first efficient algorithm to solve it. We demonstrate that, when comparing paralogous protein pairs (from Escherichia coli) of equal alignment identity and similarity, alignments that contain gaps of significant length are significantly less similar in terms of functionality, as measured with respect to Gene Ontology (GO) term similarity. This demonstrates for the first time, in a formally sound manner, that insertions and deletions cause more severe functional changes between proteins than substitutions. Our method can be reliably employed to quickly filter alignment outputs for protein pairs that are more likely to be functionally similar and/or divergent and establishes a sound and useful add-on for large-scale alignment studies.

Prokaryotic real-time gene expression profiling for toxicity assessment.

Examining global effects of toxins on gene expression profiles is proving to be a powerful method for toxicity assessment and for investigating mechanisms of toxicity. This study demonstrated the application of prokaryotic real-time gene expression profiling in Escherichia coli for toxicity assessment of environmental pollutants in water samples, by use of a cell-array library of 93 E. coli K12 strains with transcriptional green fluorescent protein (GFP) fusions covering most known stress response genes. The high-temporal-resolution gene expression data, for the first time, revealed complex and time-dependent transcriptional activities of various stress-associated genes in response to mercury and mitomycin (MMC) exposure and allowed for gene clustering analysis based on temporal response patterns. Compound-specific and distinctive gene expression profiles were obtained for MMC and mercury at different concentrations. MMC (genotoxin) induced not only the SOS response, which regulates DNA damage and repair, but also many other stress genes associated with drug resistance/sensitivity and chemical detoxification. A number of genes belonging to the P-type ATPase family and the MerR family were identified to be related to mercury resistance, among which zntA was found to be up-regulated at an increasing level as the mercury concentration increased. A mechanism-based evaluation of toxins based on real-time gene expression profiles promises, to be an efficient and informative method for toxicity assessment in environmental samples.

[Electrooptic analysis of Escherichia coli cell suspension for assessment of antibacterial activity of levomycetin].

Influence of chloramphenicol on electrophysiologic charateristics of Escherichia coli strains susceptible (K-12 strain) and resistant (pBR-325 strain) to it has been studied. It has been shown that incubation of susceptible bacteria with chloramphenicol leads to significant change of magnitute of electrooptic (EO) signal. Significant changes in orientantional spectra of suspensions of susceptible to chloramphenicol cells incubated with different concentrations of antibiotic were observed only on first five frequencies of orienting electric field (10 - 1000 kHz). Maximal change of EO signal occurred at chloramphenicol concentration 35 mg/ml and it didn't depend on the time of antibiotic exposure. Incubation of resistant strain pBR-325 with chloramphenicol did not lead to change of EO parameters of cell suspension. Potential for use of electrophysical analytic methods for assessment of antibacterial activity of chloramphenicol to control effect of antibiotics on microorganisms has been proposed.