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Inactivation kinetics of Escherichia coli K12 inoculated in blueberry, grapefruit, cantaloupe, and watermelon juices were evaluated at isothermal temperatures of 52 to 62 °C using thermal death time disks. Juices had variations in titratable acidity, pH, viscosity, and total soluble solids. Survival curves were described by Weibull and linear models, where D‐ and z‐values were determined using the first‐order model. D‐values in watermelon, cantaloupe, blueberry and grapefruit juices, were 6.57–0.64 min, 4.55–0.44 min, 3.94–0.27 min, and 3.03–0.24 min, respectively. The z‐values of E. coli K12 in tested fruit juices ranged from 5.33 to 5.89 °C. While there were no significant differences in the z‐values, the D‐values varied significantly. According to the results obtained, pH and heating temperature dramatically affect the thermal resistance of E. coli K12 under tested conditions. These findings offer a basis for developing predictive models for E. coli inactivation in fruit juices. Practical applications: Thermal death time (TDT) kinetics is extremely useful in ensuring microbial safety of fruit juices. The D‐ and z‐values calculated in this study can contribute to developing predictive models for inactivating E. coli in fruit juices with varied physicochemical attributes. Furthermore, the data and models can be used for optimization of pasteurization processes and regulatory compliance. TDT studies provide the scientific basis for process calculations and help juice manufacturers comply with regulatory requirements. Also, TDT studies help determine the minimal thermal treatments needed to inhibit spoilage organisms, ensuring the juice remains safe and palatable for longer periods. This research will help scientists understand the thermal resistance of E. coli in various fruit juice matrices. [ABSTRACT FROM AUTHOR]

The transcriptome of Esherichia coli cystlike dormant cells was investigated. RNA content in a single dormant cell was 0.26 fg, i.e., 13.5 times less than in a cell of a growing culture. The presence of mRNA in E. coli dormant cells has not been reported previously. The pools of gene reads for the dormant and growing cells were characterized, as well as the differential expression of all genes, calculated according to the special algorithm considering the average mRNA amount in a single cell. The notion of the genes active in every cell of the population was introduced. In each cell of the dormant and growing E. coli population, such genes were represented by the transcripts of 21 and 16% of the genes, respectively. The revealed cell heterogeneity regarding set of active genes is one of the reasons (and forms) of heterogeneity of bacterial populations. Sixty genes were revealed, for which activity increased twice or more during formation of E. coli dormant cells. These were the genes responsible for genome activity, structure and properties of the cell envelope, cell proliferation, stress adaptation, biofilm formation and functioning, and collective behavior, as well as the genes providing for survival of the cell population during germination of the dormant cells. [ABSTRACT FROM AUTHOR]

The release of a wide variety of persistent chemical contaminants into wastewater has become a growing concern due to their potential health and environmental risks. While the toxic effects of these pollutants on aquatic organisms have been extensively studied, their impact on microbial pathogens and their virulence mechanisms remains largely unexplored. This research paper focuses on the identification and prioritization of chemical pollutants that increase bacterial pathogenicity, which is a public health concern. In order to predict how chemical compounds, such as pesticides and pharmaceuticals, would affect the virulence mechanisms of three bacterial strains (Escherichia coli K12, Pseudomonas aeruginosa H103, and Salmonella enterica serovar. Typhimurium), this study has developed quantitative structure–activity relationship (QSAR) models. The use of analysis of variance (ANOVA) functions assists in developing QSAR models based on the chemical structure of the compounds, to predict their effect on the growth and swarming behavior of the bacterial strains. The results showed an uncertainty in the created model, and that increases in virulence factors, including growth and motility of bacteria, after exposure to the studied compounds are possible to be predicted. These results could be more accurate if the interactions between groups of functions are included. For that, to make an accurate and universal model, it is essential to incorporate a larger number of compounds of similar and different structures. [ABSTRACT FROM AUTHOR]

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism's viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium Phaeobacter inhibens DSM 17395 (Escherichia coli K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD+, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for P. inhibens DSM 17395 at ¼ ODmax were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cellsdry)−1. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria. [ABSTRACT FROM AUTHOR]

Tetrahydrofuran (THF) is a universal solvent widely used in the synthesis of chemicals and pharmaceuticals. As a refractory organic contaminant, it can only be degraded by a small group of microbes. In this study, a thiamine auxotrophic THF-degrading bacterium, Rhodococcus ruber ZM07, was isolated from an enrichment culture H-1. It was cocultured with Escherichia coli K12 (which cannot degrade THF but can produce thiamine) and/or Escherichia coli K12Δ thiE (which can neither degrade THF nor produce thiamine) with or without exogenous thiamine. This study aims to understand the interaction mechanisms between ZM07 and K12. We found that K12 accounted for 30% of the total when cocultured and transferred with ZM07 in thiamine-free systems; in addition, in the three-strain (ZM07, K12, and K12Δ thiE) cocultured system without thiamine, K12Δ thiE disappeared in the 8th transfer, while K12 could still stably exist (the relative abundance remained at approximately 30%). The growth of K12 was significantly inhibited in the thiamine-rich system. Its proportion was almost below 4% after the fourth transfer in both the two-strain (ZM07 and K12) and three-strain (ZM07, K12, and K12Δ thiE) systems; K12Δ thiE 's percentage was higher than K12's in the three-strain (ZM07, K12, and K12Δ thiE) cocultured system with exogenous thiamine, and both represented only a small proportion (less than 1% by the fourth transfer). The results of the coculture of K12 and K12Δ thiE in thiamine-free medium indicated that intraspecific competition between them may be one of the main reasons for the extinction of K12Δ thiE in the three-strain (ZM07, K12, and K12Δ thiE) system without exogenous thiamine. Furthermore, we found that ZM07 could cooperate with K12 through extracellular metabolites exchanges without physical contact. This study provides novel insight into how microbes cooperate and compete with one another during THF degradation. [ABSTRACT FROM AUTHOR]

Bacterial biofilm formation is an organized collective response to biochemical cues that enables bacterial colonies to persist and withstand environmental insults. We developed a multiscale agent-based model that characterizes the intracellular, extracellular, and cellular scale interactions that modulate Escherichia coli MG1655 biofilm formation. Each bacterium’s intracellular response and cellular state were represented as an outcome of interactions with the environment and neighboring bacteria. In the intracellular model, environment-driven gene expression and metabolism were captured using statistical regression and Michaelis-Menten kinetics, respectively. In the cellular model, growth, death, and type IV pili- and flagella-dependent movement were based on the bacteria’s intracellular state. We implemented the extracellular model as a three-dimensional diffusion model used to describe glucose, oxygen, and autoinducer 2 gradients within the biofilm and bulk fluid. We validated the model by comparing simulation results to empirical quantitative biofilm profiles, gene expression, and metabolic concentrations. Using the model, we characterized and compared the temporal metabolic and gene expression profiles of sessile versus planktonic bacterial populations during biofilm formation and investigated correlations between gene expression and biofilm-associated metabolites and cellular scale phenotypes. Based on our in silico studies, planktonic bacteria had higher metabolite concentrations in the glycolysis and citric acid cycle pathways, with higher gene expression levels in flagella and lipopolysaccharide-associated genes. Conversely, sessile bacteria had higher metabolite concentrations in the autoinducer 2 pathway, with type IV pili, autoinducer 2 export, and cellular respiration genes upregulated in comparison with planktonic bacteria. Having demonstrated results consistent with in vitro static culture biofilm systems, our model enables examination of molecular phenomena within biofilms that are experimentally inaccessible and provides a framework for future exploration of how hypothesized molecular mechanisms impact bulk community behavior. [ABSTRACT FROM AUTHOR]

A light‐addressable potentiometric sensor (LAPS) is a field‐effect‐based potentiometric device, which detects concentration changes of an analyte solution on the sensor surface in a spatially resolved way. It uses a light source to generate electron–hole pairs inside the semiconductor, which are separated in the depletion region due to an applied bias voltage across the sensor structure and hence, a surface‐potential‐dependent photocurrent can be read out. However, depending on the beam angle of the light source, scattering effects can occur, which influence the recorded signal in LAPS‐based differential measurements. To solve this problem, a novel illumination unit based on a field programmable gate array (FPGA) consisting of 16 small‐sized tunable infrared laser‐diode modules (LDMs) is developed. Due to the improved focus of the LDMs with a beam angle of only 2 mrad, undesirable scattering effects are minimized. Escherichia coli (E. coli) K12 bacteria are used as a test microorganism to study the extracellular acidification on the sensor surface. Furthermore, a salt bridge chamber is built up and integrated with the LAPS system enabling multi‐chamber differential measurements with a single Ag/AgCl reference electrode. [ABSTRACT FROM AUTHOR]

A comparative analysis of the amount and composition of the proteins adsorbed onto the surface of silver-sulfide (NpAg2S) nanoparticles obtained by biosynthesis using bacteria was carried out for the first time. These were the gram-negative bacteria Shewanella oneidensis MR-1 and Escherichia coli K12, as well as the gram-positive bacterium Bacillus subtilis 168. The biosynthesis of NpAg2S was carried out in nutrient broth with 1 mM of AgNO3 and Na2S2O3 · 5H2O salts in the presence of bacterial cells under aerobic conditions. Analysis of NpAg2S by transmission electron microscopy showed that the particles were spherical and had an average diameter of 8 ± 2 nm for S. oneidensis MR-1 and E. coli K12 and 10 ± 3 nm for B. subtilis 168. It was found that the highest amount of protein was sorbed on NpAg2S, when the strain B. subtilis 168 was used, and the smallest amount was sorbed by E. coli. The main proteins adsorbed on NpAg2S were determined by the MALDI TOF/TOF method, and the heterogeneity of the protein coating was revealed. The lowest heterogeneity of proteins on the surface of nanoparticles is observed in the case of B. subtilis (only one protein, flagellin, predominates); the highest heterogeneity of proteins was found on nanoparticles obtained using S. oneidensis MR-1. All proteins covering the surface of NpAg2S were shown to be outer-membrane or cytoplasmic-membrane proteins of the studied bacteria. The composition of the protein coating of nanoparticles is individual and constant for each bacterial strain used. The values of the ζ-potential and the effective diameter of nanoparticles were shown to differ depending on the “protein corona” of the strain that was used to obtain NpAg2S. The characteristic of the “protein corona” of biologically obtained nanoparticles is an important and necessary condition for their practical application. [ABSTRACT FROM AUTHOR]

Antibacterial properties of 15 titania photocatalysts, mono- and dual- modifi ed with nitrogen and carbon were examined. Amorphous TiO2, supplied by Azoty Group Chemical Factory Police S.A., was used as titania source (Ar-TiO2, C-TiO2, N-TiO2 and N,C-TiO2 calcined at 300°C, 400°C, 500°C, 600°C, 700°C). The disinfection ability was examined against Escherichia coli K12 under irradiation with UV and artifi cial sunlight and in dark conditions. It has been found the development of new photocatalysts with enhanced interaction ability with microorganisms might be a useful strategy to improve disinfection method conducted under artifi cial sunlight irradiation. The effi ciency of disinfection process conducted under artifi cial sunlight irradiation with carbon (C-TiO2) and carbon/nitrogen (N,C-TiO2) photocatalysts was similar as obtained under UV irradiation. Furthermore, during dark incubation, any toxicity of the photocatalyst was noted. [ABSTRACT FROM AUTHOR]

On-line monitoring of the metabolic activity of microorganisms involved in intermediate stages of biogas production plays an important role to avoid undesirable 'down times' during the biogas production. In order to control this process, an on-chip differential measuring system based on the light-addressable potentiometric sensor (LAPS) principle combined with a 3D-printed multi-chamber structure has been realized. As a test microorganism, Escherichia coli K12 ( E. coli K12) were used for cell-based measurements. Multi-chamber structures were developed to determine the metabolic activity of E. coli K12 in suspension for a different number of cells, responding to the addition of a constant or variable amount of glucose concentrations, enabling differential and simultaneous measurements. Multi-chamber differential measurement setup based on the LAPS principle; RE, reference electrode. [ABSTRACT FROM AUTHOR]

The gram-negative bacteria Escherichia coli (E. coli) is a very useful prokaryotic model for testing the toxicity of ZnO nanoparticles (nano-ZnO). This toxicity is often linked to Zn2+ released from nanoparticles in the culture medium, and nano-ZnO dissolution in different media is clearly established. Here, two model E. coli strains MG1655 and W3110 both descendant from the original K-12 showing slight differences in their genome were submitted to nano-ZnO or Zn2+ in order 1 > to refine the nano-ZnO toxicity mechanisms to E. coli, and 2 > to investigate whether toxicity resulted from a real "nanoparticle" effect or from the release of Zn2+ in solution. To do so, both strains were submitted to various concentrations (i.e., 0.1–1 mM) of nano-ZnO or Zn2+ in Luria Bertani (LB) medium. These toxicity studies take into account the nano-ZnO solubility in the culture medium by specifically monitoring the Zn2+ release in our experimental systems. In our experimental conditions, differences in tolerance to nano-ZnO or Zn2+ between both strains were clearly evidenced. W3110 is generally more tolerant to metal than MG1655, the latter showing no real difference in its sensitivity to the two zinc added forms unlike W3110. The differences in behavior between both strains could be attributed to differences in the two genomes as a mutation named "amber" in W3110. Moreover, by using these two closely E. coli strains, a real "nano" effect is here clearly demonstrated providing a model to study the toxicity of ZnO nanoparticles. [ABSTRACT FROM AUTHOR]

Escherichia coli lab strains K-12 GM4792 Lac and GM4792 Lac carry opposite lactose markers, which are useful for distinguishing evolved lines as they produce different colored colonies. The two closely related strains are chosen as ancestors for our ongoing studies of experimental evolution. Here, we describe the genome sequences, annotation, and features of GM4792 Lac and GM4792 Lac. GM4792 Lac has a 4,622,342-bp long chromosome with 4,061 protein-coding genes and 83 RNA genes. Similarly, the genome of GM4792 Lac consists of a 4,621,656-bp chromosome containing 4,043 protein-coding genes and 74 RNA genes. Genome comparison analysis reveals that the differences between GM4792 Lac and GM4792 Lac are minimal and limited to only the targeted lac region. Moreover, a previous study on competitive experimentation indicates the two strains are identical or nearly identical in survivability except for lactose utilization in a nitrogen-limited environment. Therefore, at both a genetic and a phenotypic level, GM4792 Lac and GM4792 Lac, with opposite neutral markers, are ideal systems for future experimental evolution studies. [ABSTRACT FROM AUTHOR]

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45°C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli K12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10 to 10% of the initial CFU number) surviving lethal doses of an antibiotic (ampicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term surviving persisters, and the cells of slowly growing SCV variants with small colonies ( d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete. [ABSTRACT FROM AUTHOR]

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI™ internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10³ colony-forming units (CFU) E. coli K12 ml-1, but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms. [ABSTRACT FROM AUTHOR]

The efficiency of pulsed UV light ( PL) for inactivation of E. coli K12 on hard-cooked eggs was investigated. Temperature, colour and texture were recorded to determine adverse effects on quality. Distance from the quartz window (5.5, 9.5 cm) and time (1-30 s) was set as the experimental variables. Results indicated that E. coli K12 viability decreased at closer distances and increased time ( P < 0.05). Bacterial populations were reduced to 3.54 log CFU per egg and 3.23 log CFU per egg after 15 and 20 s at 5.5 cm and 9.5 cm, respectively. Scanning electron micrographs revealed the existence of photophysical mechanisms, bacterial overlapping and internalisation interfering inactivation. Treated samples experienced slight thermal increases (6-9 °C) contributing to the preservation of colour and texture, not significantly different from untreated samples ( P > 0.05). Our findings support the use of PL to enhance the safety of hard-cooked eggs although bacterial shadowing may have significant implications on treatment efficiency. [ABSTRACT FROM AUTHOR]

Bacteria are present extensively in the environment. Investigation of their antioxidant properties will be useful for further study on atrazine stress tolerance of bacteria and the defense mechanism of antioxidant enzymes against atrazine or other triazine herbicides. Superoxide dismutase ( SOD), catalase ( CAT), glutathione S-transferase ( GST) and total antioxidant capacity ( T- AOC) from one Gram-negative representative strain Escherichia coli K12 and one Gram-positive representative strain Bacillus subtilis B19, respectively, were tested for response to atrazine stress. The results indicated that SOD, CAT, GST and T- AOC were induced upon exposure to atrazine. The growth of two bacteria was better in the absence than in the presence of atrazine, indicating that atrazine can decrease bacterial growth. The changes of enzyme activities indicate the presence of oxidative stress. Oxidative stress induced by atrazine may be due to imbalance of redox potential in bacterial cells, which leads to bacterial metabolic disorder. [ABSTRACT FROM AUTHOR]

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l with a yield of 1.02 g g total sugars during anaerobic fermentation. [ABSTRACT FROM AUTHOR]

The structure of dynamic folds in microbial chromosomes is largely unknown. Here, we find that genes with a highly biased codon composition and characterizing a functional core in Escherichia coli K12 show to be periodically distributed along the arcs, suggesting an encoded three-dimensional genomic organization helping functional activities among which are translation and, possibly, transcription. This extends to functional classes of genes that are shown to systematically organize into two independent positional gene networks, one driven by metabolic genes and the other by genes involved in cellular processing and signaling. We conclude that functional reasons justify periodic gene organization. This finding generates new questions on evolutionary pressures imposed on the chromosome. Our methodological approach is based on single genome analysis. Given either core genes or genes organized in functional classes, we analyze the detailed distribution of distances between pairs of genes through a parameterized model based on signal processing and find that these groups of genes tend to be separated by a regular integral distance. The methodology can be applied to any set of genes and can be taken as a footprint for large-scale bacterial and archaeal analysis. [ABSTRACT FROM AUTHOR]

The toxic effect of high (200 mg/l) 2,4,6-trinitrotoluene (TNT) concentrations on Escherichia coli K12 cells in the absence of exogenous nutrient sources (incubation in 0.5% NaCl) was shown to manifest itself in the transfer of the culture to an unculturable but viable state; its reversal depends on the duration of culture contact with the xenobiotic and the conditions of cell recultivation. The likelihood that cell succession to death forms the basis of the physiologo-biochemical mechanism of the unculturable state in Escherichia coli K12 population under conditions of combined toxic and starvation stress is discussed. [ABSTRACT FROM AUTHOR]

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin-resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10−4 to 10−5 log10 CFU ml g−1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10−6, log10 CFU g−1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nalR transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β-lactamase gene blaTEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid-range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic-resistant genes and the evolution of antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

The article examines whether the acidification of the cytoplasm might be the primary mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates Escherichia coli K12. According to the authors, the efficiency of DNA recovery via phenol extraction was low from K12 cells that had been subjected to HCT. They added that in cells subjected to HCT, cytoplasmic pH is reduced to a pH of 5.2, and nuclear DNA becomes entangled in coagulated cytoplasmic proteins.

The magnitude of transmembrane potential Δψ in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of Δψ in cells. In the presence of 200 mg/l of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of Δψ over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18–20 mg/l). The mean value of Δψ in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger fraction of the subpopulation with a low value of Δψ. Upon elimination of TNT, the distribution of Δψ in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics. [ABSTRACT FROM AUTHOR]

Previous reports have suggested that Escherichia coli K12 mutants defective in the expression of oligogopeptide permease protein A (OppA) exhibit reduced sensitivity to aminoglycosides due to altered permeability of the cell envelope. In this work, the role of the OppA protein, and the oligogopeptide permease (Opp) transport system has been evaluated, in the resistance to aminoglycosides using derivatives of the E. coli K12 SS320 strain selected for triornithine resistance or with a deletion of the complete opp operon. All tested mutants were defective in the uptake of tri- and tetra-peptides but did not expressed resistance to aminoglycosides. Additionally, complementation tests carried out with a plasmid encoding the OppA protein did not affect the sensitivity of the strains to these antibiotics. Taken together, these evidences indicate that the Opp uptake system, as well as the OppA protein, does not play a direct role in the sensitivity to aminoglycosides in E. coli K12. [ABSTRACT FROM AUTHOR]

Motile strains of Escherichia coli K12 carrying both a chromosomal fliC-H48 gene and a plasmid encoded fliC-H4 gene express both types of flagellins, which are coassembled into functional flagella. By using flagellar-H48-specific antiserum and a plasmid curing procedure, nonmotile mutants were found that carried an IS 5 insertion in the chromosomal fliC-H48 gene. Motile revertants were isolated that showed deletions of the IS 5 element together with sections of the fliC-H48 gene resulting in an altered flagellar serotype in these strains. As IS 5 elements were found associated with 35 of 53 known H-types in wildtype E. coli strains, this insertion element might play a major role in serotype diversity. [ABSTRACT FROM AUTHOR]

IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii. The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E. coli K12 suggest that these IS1 elements had been horizontally transferred among S. boydii and E. coli O157:H7 and K12. The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity. Analysis of the flanking sequences of IS1(SB) and its two variants in S. boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented. [Copyright &y& Elsevier]

By comparing two strains of Escherichia coli (K12 and O157:H7) with an outgroup of Salmonella and Klebsiella species and analyzing the sets of genes which are present or absent in either of the three groups, we study the gene history of K12, in particular, since the respective divergences of these bacteria. Furthermore, by using a compositional method based on context bias, we evaluate not only recently imported genes but also deleted genes. In addition, we examine recent gene duplications in the two E. coli strains. It is found that turnover of DNA is high in E. coli and, more importantly, that turnover is highest for genes of low GC content. Although levels of import are high, most of the imported genes seem to be "junk" or have poorly understood functions. Nevertheless, selected genes do persist, and may even define some E. coli strains as pathogenic. Our results support the conclusion that some of the pathogenic islands in O157:H7 are likely to have been imported in recent time. [ABSTRACT FROM AUTHOR]

The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [ Adler, J & Epstein, W. (1974) Proc. Natl Acad, Sci. USA 71, 2895- 2899] and is required for infection of the cell by bacteriophage where it most likely functions as a pore for penetration of phage DNA [ElIiott, J. & Arber, W (1 978) Mol & Gen. Genet. 161, 1-8]. The transporter consists of two transmembrane subunits (27 kDa IICMan and 31 -kDa lIDMan) and a hydrophilic subunit (35-kDa lIABMan). Protein fusions of IICMan and IIDMan with β-gaIaetosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins Protein fusions were obtained by progressively deleting the manY and manZ genes from their ends and ligating them to manZ and phoA that lack promotor and leader sequences. Based on the analysis of 30 HCMan PhoA, 10 IICMMan LacZ, 12 IIDMan- PhoA, and 30 lIDMan LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane. lIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm. [ABSTRACT FROM AUTHOR]

Several growth monitoring parameters, including adenine nucleotide contents, were measured during Escherichia coli K12 batch cultivation in mineral medium with glucose. The adenylate energy charge with its mean value of 0.83 remained roughly stable during growth. The total adenylate and ATP pools (nmol/mg dry weight), and also the individual cell volume changed with a pattern of two maxima at approximately 4 and 10 h of cultivation. After the exhaustion of the glucose from the growth medium the adenylate energy charge, the pools of ATP and total adenylate started to decrease marking the onset of the stationary growth phase. Our data indicate that actually there was only a limited period within the logarithmic growth phase during which the growth might have been balanced: during this period of 1.5 h different growth monitoring parameters (optical absorbance, cell number, total cell volume, and ATP content per ml) increased with almost equal rates. Moreover, as ATP pool and median cell volume during this short period were approximately constant, the culture might have been even in the steady state. [ABSTRACT FROM AUTHOR]

The uptake of methyl α- d-glucopyranoside (α-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells. The inhibition, which occurred only at non-saturating concentrations of α-MG, was not the result of a stimulation of the rate of exit of intracellular α-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide. Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of α-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration. [ABSTRACT FROM AUTHOR]

The contiguous 874.423 base pair sequence corresponding to the 50.0–68.8 min region on the genetic map of the Escherichia coli K-12 (W3110) was constructed by the determination of DNA sequences in the 50.0–57.9 min region (360 kb) and two large (100 kb in all) and five short gaps in the 57.9–68.8 min region whose sequences had been registered in the DNA databases. We analyzed its sequence features and found that this region contained at least 894 potential open reading frames (ORFs), of which 346 (38.7%) were previously reported, 158 (17.7%) were homologous to other known genes, 232 (26.0%) were identical or similar to hypothetical genes registered in databases, and the remaining 158 (17.7%) showed no significant similarity to any other genes. A homology search of the ORFs also identified several new gene clusters. Those include two clusters of fimbrial genes, a gene cluster of three genes encoding homologues of the human long chain fatty acid degradation enzyme complex in the mitochondrial membrane, a cluster of at least nine genes involved in the utilization of ethanolamine, a cluster of the secondary set of 11 hyc genes participating in the formate hydrogenlyase reaction and a cluster of five genes coding for the homologues of degradation enzymes for aromatic hydrocarbons in Pseudomonas putida. We also noted a variety of novel genes, including two ORFs, which were homologous to the putative genes encoding xanthine dehydrogenase in the fly and a protein responsible for axonal guidance and outgrowth of the rat, mouse and nematode. An isoleucine tRNA gene, designated ileY , was also newly identified at 60.0 min. [ABSTRACT FROM PUBLISHER]

The 465,813 base pair sequence corresponding to the 40.1–50.0 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. Analysis of the sequence revealed that this region contained at least 466 potential open reading frames, of which 187 (40%) were previously reported, 105 (23%) were homologous to other known genes, 103 (22%) were identical or similar to hypothetical genes registered in databases, and the remaining 71 (15%) did not show a significant similarity to any other gene. At the 45.2–46.0 min region, we found a very large cluster of about 30 genes, whose functions are involved in the biosynthesis of polysaccharides as the components of outer membranes. In addition, we identified anew asn-tRNA gene, designated asnW, between the asnT and asnU genes and a new lysogenic phage attachment site as the cis-element. [ABSTRACT FROM PUBLISHER]

The 569,750 base pair sequence corresponding to the 28.0–40.1 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. This region includes the replication terminus region and contained at least 549 potential open reading frames. Among them, 160 (29%) were previously reported, 174 (32%) were homologous to other known genes, 102 (18%) were identical or similar to hypothetical genes registered in databases, and the remaining 113 (21%) did not show a significant similarity to any other gene. Of interest was the finding of a large number of genes and gene clusters in andnear the replication termination region which had been thought to be genetically silent. Those includeda cluster of genes for fatty acid β-oxidation, the third copy of the pot (spermidine/putrescine transport system) gene cluster, the second dpp (dipeptide transport system) operon, the second dsm (anaerobic dimethyl sulfoxide reductase) operon, a cluster of fim (fimbrial) genes anda DNA helicase-like gene with a high molecular weight. In addition, we found the dnaC- and dnaT-like genes in the cryptic prophage, Rac, anda number of genes originated probably from plasmids. [ABSTRACT FROM PUBLISHER]

The 718,122 base pair (bp) sequence of the Escherichia coli K-12 genome corresponding to the region from 12.7 to 28.0 minutes on the genetic map is described. This region contains at least 682 potential open reading frames, of which 278 (41%) have been previously identified, 147 (22%) were homologous to other known genes, 138 (20%) are identical or similar to the hypothetical genes registered in databases, and the remaining 119 (17%) did not show a significant similarity to any other gene. In this region, we assigned a cluster of cit genes encoding multienzyme citrate lyase, two clusters of fimbrial genes and a set of lysogenic phage genes encoding integrase, excisionase and repressor in the e14 genetic element. In addition, a new valine tRNA gene, designated valZ, and a family of long directly repeated sequences, LDR-A, -B and -C, were found. [ABSTRACT FROM PUBLISHER]

Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to ≤0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145000 Da (± 10000 Da), judged by gel-filtration chromatography. Subunits of 73000 Da ( ± 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30°C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 μM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 μM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (Ki = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 μtM at 10 μM thiamine diphosphate) than with other divalent cations. [ABSTRACT FROM AUTHOR]