Polynucleotide Sequence Divergence Among Strains of Escherichia coli and Closely Related Organisms

Polynucleotide sequence similarity tests were carried out to determine the extent of divergence present in a number of Escherichia coli strains, obtained from diverse human, animal, and laboratory sources, and closely related strains of Shigella, Salmonella, and the Alkalescens-Dispar group. At 60 C, relative reassociation of deoxyribonucleic acid (DNA) from the various strains with E. coli K-12 DNA ranged from 100 to 36%, with the highest level of reassociation found for three strains derived from K-12, and the lowest levels for two “atypical” E. coli strains and S. typhimurium. The change in thermal elution midpoint, which indicates the stability of DNA duplexes, ranged from 0.1 to 14.5 C, with thermal stability closely following the reassociation data. Reassociation experiments performed at 75 C, at which temperature only the more closely related DNA species form stable duplexes, gave similar indications of relatedness. At both temperatures, Alkalescens-Dispar strains showed close relatedness to E. coli, supporting the idea that they should be included in the genus Escherichia. Reciprocal binding experiments with E. coli BB, 02A, and K-12 yielded different reassociation values, suggesting that the genomes of these strains are of different size. The BB genome was calculated to be 9% larger than that of K-12, and that of 02A 9% larger than that of BB. Calculation of genome size for a series of E. coli strains yielded values ranging from 2.29 × 10⁹ to 2.97 × 10⁹ daltons. E. coli strains and closely related organisms were compared by Adansonian analysis for their relatedness to a hypothetical median strain. E. coli 0128a was the most closely related to this median organism. In general, these data compared well with the data from reassociation experiments among E. coli strains. However, anomalous results were obtained in the cases of Shigella flexneri, S. typhimurium, and “atypical” E. coli strains.     

Antibody Profiling of Bipolar Disorder Using Escherichia coli Proteome Microarrays

To profile plasma antibodies of patients with bipolar disorder (BD), an E. coli proteome microarray comprising ca. 4200 proteins was used to analyze antibody differences between BD patients and mentally healthy controls (HCs). The plasmas of HCs and patients aged 18–45 years with bipolar I disorder (DSM-IV) in acute mania (BD-A) along with remission (BD-R) were collected. The initial samples consisting of 19 BD-A, 20 BD-R, and 20 HCs were probed with the microarrays. After selecting protein hits that recognized the antibody differences between BD and HC, the proteins were purified to construct BD focus arrays for training diagnosis committees and validation. Additional six BD-A, six BD-R, six HCs, and nine schizophrenic disorder (SZ, as another psychiatric control) samples were individually probed with the BD focus arrays. The trained diagnosis committee in BD-A versus HC combined top six proteins, including rpoA, thrA, flhB, yfcI, ycdU, and ydjL. However, the optimized committees in BD-R versus HC and BD-A versus BD-R were of low accuracy (< 0.6). In the single blind test using another four BD-A, four HC, and four SZ samples, the committee of BD-A versus HC was able to classify BD-A versus HC and SZ with 75% sensitivity and 80% specificity that both HC and SZ were regarded as negative controls. The consensus motif of the six proteins, which form the committee of BD-A versus HC, is [KE]DIL[AG]L[LV]I[NL][IC][SVKH]G[LV][VN][LV] by Gapped Local Alignment of Motifs. We demonstrated that the E. coli proteome microarray is capable of screening BD plasma antibody differences and the selected proteins committee was successfully used for BD diagnosis with 79% accuracy.The etiology and genetic contributions of bipolar disorder (BD)¹ largely remain unknown (1). Because of the presumed high level of etiologic heterogeneity and the overlap of dimensions across mood disorders and schizophrenia (2), the main difficulty in making an exact diagnosis for psychiatric disorder is the lack of pathological biochemical index (3). However, several lines of evidence support that various immunomodulatory factors, such as cytokine and soluble cytokine receptor, play an integral role in the pathophysiology of bipolar disorder (4–7). For example, several studies have reported that cell-mediated immunity cytokine abundance is correlated with mood state (8, 9). Our early works also found that higher levels of soluble interleukin-2 receptor (sIL-2R) (5, 10) and interleukin 1 receptor antagonist (IL-1Ra) (5, 11) are accompanied with bipolar mania. Furthermore, the abnormalities of total immunoglobulins levels in body fluid are observed in BD patients (12, 13).The possibility of biomarkers for assisting BD diagnosis has been recently highlighted (14–16). Tumor necrosis factor alpha (TNF-α), 3-nytrotrosine, interleukin-6, interleukin-10, and brain-derived neurotrophic factor in body fluids are potentially useful for classifying stages of BD (15). Nevertheless, they are not specific for distinguishing from other psychiatric diseases (17). Chronic inflammation exists in medicated bipolar patients displaying varied correlations with leptin, insulin, soluble TNF receptor-1 (sTNF-R1), and IL-1Ra (11). Notwithstanding, controversy exists as to whether these phenomena are state-dependent (5), normalize in remission (18), or represent trait markers exacerbated by the affective episodes (19). These discrepancies may be explained by heterogeneity in mood state, methodological differences, and not controlling for known confounds, such as obesity (6). In addition to inflammatory markers, increasing production of antibodies (20–22) and immunoglobulins (23, 24) may be implicated with BD.In recent years, proteomic technologies based on mass spectrometry have been increasingly used, especially in the search for diagnostic and prognostic biomarkers in neuropsychiatric disorders (25). Protein microarrays have been demonstrated as an effective high throughput platform for analysis of aberrant immune responses in diseases (26–29). It is hypothesized that the trait or state-dependent biomarkers of bipolar disorder may exist. We attempted to identify a committee of proteins for the diagnosis of BD through employing the ca. 4200 E. coli proteins in a microarray format. The two-phase strategy for identification and validation protein hits (30) was used in this study. Although the antigens on the microarray may not be directly associated with BD, this microarray provided hundreds of thousands of epitopes for analyzing antibody profiles of plasma samples in a high throughput fashion.     

Divergence and redundancy of transport and metabolic rate-yield strategies in a single Escherichia coli population

The energetic efficiency of nutrient uptake and conversion into biomass is a key factor in the ecological behavior of microorganisms. The constraints shaping the metabolic rate-yield trade-off in bacteria are not well understood. To examine whether metabolic rate-yield settings and physiological strategies evolve toward a particular optimum in a constant environment, we studied multiple Escherichia coli isolates evolving in a glucose-limited chemostat population. A major divergence in transport and metabolic strategies was observed, and the isolates included inefficient rate strategists (polluters or cheaters) and yield strategists (conservationists), as well as various hybrid rate-yield strategists and alternative ecotypes (dropouts). Sugar transport assays, strain comparisons based on metabolomics, and Biolog profiling revealed variance to the point of individuality within an evolving population. Only 68 of 177 metabolites assayed were not affected in 10 clonally related strains. The parallel enrichment of rate and yield strategists and the divergence in metabolic phylogenies indicate that bacteria do not converge on a particular rate-yield balance or unique evolutionary solutions. Redundancies in transport and metabolic pathways are proposed to have laid the framework for the multiplicity of bacterial adaptations.     

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10⁻⁵. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.     

Heterogeneity of Escherichia coli population during induced autolysis

Escherichia coli M-17 autolysis was induced by eliminating nutrition sources from the growth medium and exerting a shock with EDTA. The overall cell number, the optical density of the cell suspension, the number of colony-forming units (CFU), and [3H]uracil incorporation into the cells were analysed in the course of autolysis. The number of CFU was found to drop down faster than the overall cell number in the process of autolysis. The population of E. coli was shown to be heterogeneous in its sensitivity to the induction of autolysis, and some nonlysed cells were still metabolically active. When the rate of autolysis was highest in some cells of the population, the labeled precursor was found to be incorporated into the TCA-soluble and TCA-insoluble fractions of nonlysed cells. The overall cell number, the optical density of the cell suspension, and the number of CFU increased 96 h after the induction of autolysis. The authors discuss what is the role played by the heterogeneity of an E. coli population in its adaptation to EDTA-induced autolysis.     

Plasmolysis during the division cycle of Escherichia coli

Cells of Escherichia coli were plasmolyzed with sucrose. They were classified according to length by way of electron micrographs taken from samples prepared by agar filtration. The percentage of plasmolyzed cells increased about two- and threefold between mean cell sizes of newborn and separating cells. However, dividing cells were less frequently plasmolyzed than nondividing cells of the same length class. Analysis of cell halves (prospective daughters) in dividing cells showed that they behaved as independent cellular units with respect to plasmolysis. The results indicate that compressibility of the protoplast (given a certain plasmolysis space) is inversely related to cell size. That a dividing cell does not react as one osmotic compartment to osmotic stress may suggest that cell size-dependent strength of the cell membrane-cell wall association, rather than variation in turgor, plays a role during the cell division cycle.     

Fate of Escherichia coli during Ensiling of Wheat and Corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 × 10⁸ CFU/g into direct-cut wheat (348 g of dry matter kg⁻¹), wilted wheat (450 g of dry matter kg⁻¹), and corn (375 g of dry matter kg⁻¹). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

Fate of Escherichia coli during ensiling of wheat and corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 x 10(8) CFU/g into direct-cut wheat (348 g of dry matter kg(-1)), wilted wheat (450 g of dry matter kg(-1)), and corn (375 g of dry matter kg(-1)). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

Significance of rpoS during maturation of Escherichia coli biofilms

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.     

Lipid synthesis during the Escherichia coli cell cycle.

Lipid synthesis was examined in Escherichia coli cells at different stage of cell division. Exponentially growing cells were pulse-labeled with appropriate isotopes for 0.1 generation time, inactivated, and separated by size on a sucrose gradient. An abrupt increase in the rate of lipid synthesis occurred which was coincident with the initiation of cross walls. In contrast, the rate of protein synthesis during this same interval remained constant, resulting in an increased lipid/protein ratio in dividing cells. No changes in the composition of phospholipid head groups, fatty acids, or phospholipid molecular species were observed in cells at different stages of division. The observed increase in the rate of lipid synthesis may reflect a means by which the activities of membrane-associated enzymes are modulated during cross wall formation.     

Loss of plasmids during enrichment for Escherichia coli.

Enrichment with sodium lauryl sulfate and incubation at 44.5 degrees C resulted in a loss of plasmids and decreased efficiency in the recovery of pathogenic. Escherichia coli strains from foods.     

Cell division during nutritional upshifts of Escherichia coli.

Nutritional shifts of Escherichia coli B/r to richer media have been analyzed is synchronously growing and exponential-hase populations. Early perturbations in the timing of cell division were observed. At the slow growth, division progressed at a rate equal to or less than the preshift rate for about 1 h. At intermediate growth, both delays and acceleration in division were observed. The extent of the perturbation depended upon the age of the cells at the time of the shift and the composition of the preshift and postshift media. The perturbation was different in the two substrains of E. coli B/r used in this study.