Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

Transport and utilizing of the biosynthetic intermediate shikimic acid in Escherichia coli

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poort growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced.Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport.Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% contransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not contransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that bot classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (K_m) for shikimate transport.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide Riboside by Escherichia coli

The acculnulation of 5 (4) -amino-4 (5) -imidazolecarboxamide riboside (AICA-R) in the culture medium of sulfonamide-inhibited Escherichia coli, and E. coli-like bacteria was studied. E. coli strain Band 32 strains of E. coli-like bacteria accumulated more than 50 μmoles of AICA-R in test tube scale experiments, and one of E. coli-like bacteria accumulated 358 μmoles. E. coli B-96 (purine-requiring mutant) had ability to accumulate AICA-R in the glucose-salt medium containing purine bases, especially xanthine. The addition of glycine alone or together with glutamic acid to the glucose-salt medium increased the accumulation of AICA-R by sulfadiazine-inhibited E. coli strain B. The accumulation was considerably increased by the addition of polypeptone or casein hydrolysate.

AICA-R accumulated during sulfadiazine bacteriostasis of E. coli strain B was purified and crystallized according to the procedure of Greenberg and Spilman, and light amber colored crystals were obtained.     

Participation of RNase I in MS2 Phage Growth

Participation of RNase I in the growth of phage on infection with bacteriophage MS2 was studied.

Some strains of uracil-requiring E. coli were isolated, grown in MS broth, and transferred to a minimal medium to exhaust the pool of nucleotides. The phage was then added to the cells grown in uracil-deficient medium. The growth of phage was observed to occur at the burst size of two hundreds in strains of E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, which possessed RNase I, but not in strains, A19 (Hfr) U^? and Q13 (Hfr) U^?, which lacked RNase I.

A marked increase in acid-soluble fraction was observed with E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, whereas the increase was little with E. coli A10 (Hfr) U^? and Q13 (Hfr) U^? Conditions for the growth of phage in uracil-deficient medium were investigated and the effect of antibiotics were also investigated.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

Summary We have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b).In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.     

Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K‐12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10²–10⁷ colony‐forming units (CFU) in E. coli and 10³–10⁷ CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real‐time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL‐producing system of P. luminescens appeared to be quite unstable. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of cultural conditions and mutations on the composition of the outer membrane proteins ofEscherichia coli

Summary VariousEscherichia coli strains differ in the composition of their major outer membrane proteins. However, allE. coli K12 strains tested possess the same major outer membrane proteinsa, b, c andd although quantitative differences were detected.The influence of growth conditions on the composition of the major outer membrane proteins ofE. coli was analyzed. It was found that neither the growth phase at which the cells are harvested, nor the fatty acid composition of the phospholipids has a considerable influence on the composition of these proteins. However, the composition of the growth medium, and, to a less extent, the growth temperature, have a pronounced influence.Certain mutants, changed in the composition of their lipopolysaccharide, are deficient in proteinb. Also mutants deficient in proteinc andd respectively, are described.Proteinsb andc ofE. coli K12 were found to be associated with peptidoglycan. Protein bands, corresponding with flagellin and pilin respectively, were identified.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazole- carboxamide by Escherichia coli in a Shaking Culture

Various attempts were made to accumulate 5 (4) -amino-4 (5) -imidazolecarboxamide (abreviated as AICA; in this paper, separate analyses for the riboside forms were not attempted, and hence AICA and AICA-riboside will be presented as total AICA) in a shaking culture medium by Escherichia coli strain B. The accumulation of non-acetylatable, diazotizable amines was accomplished by the addition of 0.01% of sulfadiazine, 0.2% of glucose, and 2% of peptone in the medium for sixteen hours at 30°C. E coli strain B was able to accumulate the amines in the pepton medium, even when glucose and the sulfonamide inhibtor were omitted. Although paper chromatographic and spectrophotometric analyses proved the accumulation of AICA and AICA-riboside by E. coli train B in the medium, another substance colored by the Bratton and Marshall method was also accumulated.     

Laboratory adapted Escherichia coli K‐12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis

Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K‐12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K‐12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human‐pathogenic E. coli infectious disease.     

99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

PurposeRadiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.Methods and resultsIdentical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were ^99mTc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of ^99mTc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8 pmol, respectively, per 10¹⁰ cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90 min for both ^99mTc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of ^99mTc-study MORF was significantly higher (p = 0.009) than that of the control in the thigh with sterile inflammation.ConclusionA ^99mTc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.     

Survival of Escherichia coli during drying and storage in the presence of compatible solutes

Five different compatible solutes, sucrose, trehalose, hydroxyectoine, ectoine, and glycine betaine, were investigated for their protective effect on Escherichia coli K12 and E. coli NISSLE 1917 during drying and subsequent storage. Two different drying techniques, freeze-drying and air-drying, were compared. The highest survival rate was observed when the non-reducing disaccharides sucrose (for E. coli K12) and trehalose (for E. coli NISSLE 1917) were added. The two tetrahydropyrimidines, hydroxyectoine and ectoine, gave protection to freeze-dried E. coli NISSLE 1917 whereas E. coli K12 was protected only by hydroxyectoine. Glycine betaine seemed to be harmful for both strains of E. coli with both drying techniques. Air0drying gave much better survival rates than freeze-drying. The two strains of E. coli differed in their ability to take up compatible solutes.     

Marine macroalgae as a source for osmoprotection for Escherichia coli

At elevated osmolarity of the mineral medium M63, marine macroalgae constitute important osmoprotectants and nutrients sources for Escherichia coli. Growth of bacterial population (16 strains) was improved by supplementing M63 salts medium with either aqueous or ethanolic algal extracts obtained from Ascophyllum nodosum, Fucus serratus, Enteromorpha ramulosa, Ulva lactuca, and Palmaria palmata. In their presence, growth was still observed even at 1.02 m NaCl. Furthermore, the E. coli ZB400 growth in presence of whole macroalgae thalli in M63/0.85 m NaCI reached its maximum within 24 h (5 × 10⁷ – 5 × 10⁸ colony-forming units [CFU] per milliliter). In the presence of A. nodosum, bacterial growth was inhibited. In the same experimental conditions, ethanolic extracts improved E. coli growth significantly, because the yield reached 10¹¹ CFU per milliliter. Ulva lactuca and P. palmata allowed the better growth. The Dragendorff-positive compounds extracted from bacterial cells growing on each ethanolic extract exhibited an osmoprotective effect as proved by a disk-diffusion assay. On the other hand, the -onium compounds (quaternary ammonium [betaines] and tertiary sulphonium) and total free amino acid contents of U. lactuca ethanolic extracts were higher than in others. Fucaceae extracts demonstrated especially high protein content. Algal extracts constitute not only an appreciable osmoprotection source for E. coli but also nutrient sources. Correspondence to: J. Minet     

Comparison of acetate inhibition on growth of host and recombinantE. coli K12 strains

Summary We investigated the extent of acetate inhibition on growth rate of different host and recombinantE. coli K12 strains, cultivated in defined and complex media. A mathematical model was proposed to describe this inhibition. Our results show that acetate inhibition is more significant for recombinant cells than for host cells, and for cells cultured in defined medium than in complex medium.     

Breakdown ofd-glucose by mixed cultures ofEscherichia coli,Desulfovibrio vulgaris,andChromatium vinosum

Experiments with mixed cultures ofEscherichia coli, Desulfovibrio vulgaris, andChromatium vinosum were conducted using a synthetic medium with glucose as the substrate. The bacterial number and the changes in the chemical factors were determined during the development of the mixed culture. (i) Fermentation of glucose byE. coli produces organic acids (formic, acetic, lactic, and succinic) and alcohol. The growth-yield constant K (cell dry weight per weight solid substrate) does not exceed 0.05. (ii) In the mixed culture ofE. coli andD. vulgaris, the reduction of sulfate is accompanied by a total consumption of formate, lactate, and alcohol and an increase in the sulfide and acetate content. (iii) When the three physiologically different species are allowed to grow simultaneously, there is no accumulation of catabolites in the medium and the growth yield constant increases to 0.46. Maximum phototrophic production requires the presence of bothE. coli andD. vulgaris. A low substrate concentration and the simultaneous growth of the three organisms are other factors that contribute towards a high output. The biochemical parameters of the medium are influenced to a large extent by the glucose level. The results suggest that the behavior of the strains is different in pure and mixed cultures.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Role of nutrient limitation in the competition between uropathogenic strains of Klebsiella pneumoniae and Escherichia coli in mixed biofilms

Klebsiella pneumoniae and Escherichia coli form mixed species biofilms in catheter-associated urinary tract infections. Recently, a detrimental effect of K. pneumoniae over E. coli was observed in mixed species biofilms grown in an artificial urine medium. The mechanism behind this competitive interaction was studied. K. pneumoniae partially outcompeted E. coli in early-stage batch-fed biofilms, whereas both microorganisms co-exist at longer times (K. pneumoniae:E. coli ratio, 55:1), as shown by cell counts and confocal microscopy. E. coli cells were scattered along the K. pneumoniae biofilm. Biofilm supernatants did not appear to contain either antimicrobial or anti-biofilm activities against E. coli. Biofilms grown under continuous flow prevented interspecies competition. K. pneumoniae showed both increased siderophore production and better growth in iron-limited media compared to E. coli. In summary, these results indicate the importance of nutrient (particularly iron) competition in the modulation of the bacterial composition of mixed species biofilms formed by uropathogenic K. pneumoniae and E. coli.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Adhesion of enterotoxigenic Escherichia coli to immobolized intestinal mucosal preparations: a model for adhesion to mucosal surface components

The ability of enterotoxigenic strains of E. coli to adhere to immobilized mucosal components prepared from the large and small intestines of mice was examined in vitro. Various strains of E. coli were labeled with (³H)-acetate and incubated in tissue culture plates containing immobilized mucosal components or bovine serum albumin. E. coli strains which were positive for K88 or K99 antigen, and one E. coli strain isolated from a human urinary tract infection, were shown to adhere readily to large and small intestinal mucosal preparations, but not to bovine serum albumin. E. coli K-12 and a variety of enterotoxigenic strains isolated from humans, including a CFA/1 positive strain, demonstrated little or no ability to adhere to any of the preparations. E. coli adhesion to the mucosal preparations was shown to be mannose-resistant for all E. coli strains tested, but was inhibited by growth of the organisms at 18°C. Adhesion of each of the K88 or K99 positive strains was inhibited by homologous antiserum, but not by heterologous antiserum or normal rabbit serum. The data indicate that the mucosal preparations employed contain receptors for specific bacterial adhesins, and suggest that the use of such preparations may provide an alternative to the use of whole cells both as a source of receptor and as a means of investigating the adhesive properties of E. coli.     

Isolation and characterization of mutants ofEscherichia coli defective in pyridine nucleotide cycle enzymes

Thirty-eight analogs of nicotinic acid and nicotinamide were tested for their ability to inhibit growth of wild-typeEscherichia coli K-12. Two of the compounds tested, 6-aminonicotinic acid and 6-aminonicotinamide were strongly inhibitory to growth of the organism. Mutants resistant to these compounds were isolated and characterized by cross-feeding experiments. All of the mutants isolated by their resistance to these analogs were found to excrete a metabolite which supported growth ofnadA, nadB, ornadC strains ofE. coli on a minimal medium. Wild-type strains failed to exhibit this cross-feeding ability. ThepncB ⁺ locus codes for nicotinic acid phosphoribosyl transferase and maps near minute 23 on the chromosome.