SPECIFIC DELETION OCCURRING IN THE DIRECTED EVOLUTION OF 6-PHOSPHOGLUCONATE DEHYDROGENASE IN ESCHERICHIA COLI

ABSTRACT A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E. coli has been observed. The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS 5) found normally in E. coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map. The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate.

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Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

Applied and Environmental Microbiology ◽

10.1128/aem.70.8.4499-4504.2004 ◽

2004 ◽

Vol 70 (8) ◽

pp. 4499-4504 ◽

Cited By ~ 49

Author(s):

Manuel Ferrer ◽

Tatyana N. Chernikova ◽

Kenneth N. Timmis ◽

Peter N. Golyshin

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Growth Temperature ◽

Specific Activity ◽

Maximum Growth ◽

Maximum Growth Rate ◽

Coli Strain ◽

Temperature Sensitive ◽

Psychrophilic Bacterium ◽

E Coli

ABSTRACT A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h−1) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min−1 g−1). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).

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Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach

Microbiology ◽

10.1099/mic.0.26560-0 ◽

2004 ◽

Vol 150 (5) ◽

pp. 1413-1426 ◽

Cited By ~ 71

Author(s):

Robert A. Cox

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Mycobacterium Tuberculosis ◽

Growth Rates ◽

Specific Growth ◽

Streptomyces Coelicolor ◽

Maximum Growth ◽

Maximum Growth Rate ◽

E Coli ◽

Specific Growth Rates

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 °C, and Escherichia coli B/r, grown at 37 °C. A frame of reference was established based on quantitative relationships observed between specific growth rates (μ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally (μ=0·30 h−1) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli (μ=1·73 h−1); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating μ to the number of rrn operons per genome. Values of μ=0·69 h−1 and μ=1·00 h−1 were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

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Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.608839 ◽

2021 ◽

Vol 12 ◽

Author(s):

Almaz Nigatu Tesfahun ◽

Marina Alexeeva ◽

Miglė Tomkuvienė ◽

Aysha Arshad ◽

Prashanna Guragain ◽

...

Keyword(s):

Escherichia Coli ◽

Excision Repair ◽

Dna Lesions ◽

Base Pairs ◽

E Coli ◽

Thymine Glycol ◽

Base Excision ◽

The Cost ◽

Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

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Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

Infection and Immunity ◽

10.1128/iai.01329-12 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2733-2742 ◽

Cited By ~ 26

Author(s):

Alexandre Bleibtreu ◽

Pierre-Alexis Gros ◽

Cédric Laouénan ◽

Olivier Clermont ◽

Hervé Le Nagard ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Stress Response ◽

Mouse Model ◽

Stress Resistance ◽

Maximum Growth ◽

Competitive Fitness ◽

Content Type ◽

General Stress Response ◽

E Coli

ABSTRACTThe extraintestinal virulence ofEscherichia coliis dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuringin vitroindividual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenicE. colistrains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescentE. colistrain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2resistance, andrpoSsequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.

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Antimicrobial Effect of Picea abies Extracts on E. coli Growth

Molecules ◽

10.3390/molecules24224053 ◽

2019 ◽

Vol 24 (22) ◽

pp. 4053 ◽

Cited By ~ 2

Author(s):

Nabil Haman ◽

Ksenia Morozova ◽

Giustino Tonon ◽

Matteo Scampicchio ◽

Giovanna Ferrentino

Keyword(s):

Picea Abies ◽

Essential Oils ◽

Isothermal Calorimetry ◽

Antimicrobial Effect ◽

Maximum Growth ◽

Lag Time ◽

Maximum Growth Rate ◽

Plate Count ◽

E Coli ◽

Wood Residues

This study aims to investigate the effect of essential oils extracted from wood residues of Picea abies on the growth of Escherichia coli. The essential oils were extracted by supercritical carbon dioxide, leading to a yield of 3.4 ± 0.5% (w/w) in 120 min. The antimicrobial effect was tested at 37 °C by isothermal calorimetry. The heat-flow (dq/dt vs. time) was integrated to give a fractional reaction curve (α vs. time). Such curves were fitted by a modified Gompertz function to give the lag-time (λ) and the maximum growth rate (µmax) parameters. The results showed that λ was linearly correlated with E. coli concentration (λ = 1.4 h/log (CFU/mL), R2 = 0.997), whereas µmax was invariant. Moreover, the overall heat was nearly constant to all the dilutions of E. coli. Instead, when the essential oil was added (with concentrations ranging from 1 to 5 mg/L) to a culture of E. coli (104 CFU/mL), the lag-time increased from 14.1 to 33.7 h, and the overall heat decreased from 2120 to 2.37 J. The results obtained by the plate count technique were linear with the lag-time (λ), where (λ = −7.3 × log (CFU/mL) + 38.3, R2 = 0.9878). This suggested a lower capacity of E. coli to metabolize the substrate in the presence of the essential oils. The results obtained in this study promote the use of essential oils from wood residues and their use as antimicrobial products.

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Cloning of promoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11 and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillus reuteri

Canadian Journal of Microbiology ◽

10.1139/m94-165 ◽

1994 ◽

Vol 40 (12) ◽

pp. 1043-1050 ◽

Cited By ~ 17

Author(s):

Gordana Djordjevic ◽

Bojana Bojovic ◽

Ana Banina ◽

Ljubisa Topisirovic

Keyword(s):

Escherichia Coli ◽

Lactococcus Lactis ◽

Dna Sequences ◽

Lactobacillus Reuteri ◽

Lactobacillus Paracasei ◽

Translational Efficiency ◽

Broad Host Range ◽

Chromosomal Dna ◽

Base Pairs ◽

E Coli

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5′ end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative −35 and −10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between these two hexanucleotides and between the putative −10 hexanucleotide and the transcriptional start point (A residues predominated) ranged from 17 to 18 base pairs and from 5 to 7 base pairs, respectively. Each of the cloned Lactobacillus paracasei CG11 promoter-like fragments contained an AT-rich sequence upstream of the putative −35 region (from 60 to 73%).Key words: Lactobacillus, promoter-like sequences, transcriptional efficiency, translational efficiency.

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Growth of Stressed Strains of Four Non-O157 Shiga Toxin–Producing Escherichia coli Serogroups in Five Enrichment Broths

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-019 ◽

2015 ◽

Vol 78 (11) ◽

pp. 1960-1966 ◽

Cited By ~ 4

Author(s):

BAVO VERHAEGEN ◽

KOEN DE REU ◽

MARC HEYNDRICKX ◽

INGE VAN DAMME ◽

LIEVEN DE ZUTTER

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Growth Dynamics ◽

Maximum Growth ◽

Maximum Growth Rate ◽

Lag Phase ◽

Sodium Pyruvate ◽

Peptone Water ◽

Pure Cultures ◽

Freeze Stress

The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, −20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila's enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

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IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations.

Genetics ◽

10.1093/genetics/121.3.423 ◽

1989 ◽

Vol 121 (3) ◽

pp. 423-431 ◽

Cited By ~ 2

Author(s):

B G Hall ◽

L L Parker ◽

P W Betts ◽

R F DuBose ◽

S A Sawyer ◽

...

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Insertion Sequence ◽

Natural Populations ◽

Single Copy ◽

Target Sequence ◽

Wild Type ◽

Insertion Element ◽

E Coli ◽

Natural Isolates

Abstract IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.

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Characterization and Complete Genome Analysis of a Novel Escherichia Phage, vB_EcoM-RPN242

10.21203/rs.3.rs-1207197/v1 ◽

2022 ◽

Author(s):

Napakhwan Imklin ◽

Pattaraporn Sripras ◽

Narut Thanantong ◽

Porntippa Lekcharoensuk ◽

Rujikan Nasanit

Keyword(s):

Escherichia Coli ◽

Nucleotide Sequence ◽

Sequence Comparison ◽

The Novel ◽

Base Pairs ◽

E Coli ◽

Double Stranded Dna ◽

Protein Encoding ◽

Encoding Genes ◽

Complete Genome Analysis

Abstract The novel Escherichia phage vB_EcoM-RPN242 was isolated using a strain of Escherichia coli host originated from a diarrheal piglet. The phage was able to form plaques on the E. coli lawn at 15−45ºC. Moreover, it was stable over a wide pH (4−10) and temperature (4−70ºC) range. The vB_EcoM-RPN242 genome was found to be a linear, double-stranded DNA consisting of 154,840 base pairs. There were 195 protein-encoding genes and 2 tRNAs detected in the genome, however no unfavorable gene was found. According to the overall nucleotide sequence comparison, the vB_EcoM-RPN242 possibly represents a new phage species in the genus Agtrevirus.

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Enzyme Histochemistry of the Small Intestinal Mucosa in Experimental Infections of Calves with Rotavirus and Enterotoxigenic Escherichia coli

Veterinary Pathology ◽

10.1177/030098588602300204 ◽

1986 ◽

Vol 23 (2) ◽

pp. 125-131 ◽

Cited By ~ 4

Author(s):

M. T. Stiglmair-Herb ◽

A. Pospiscihl ◽

R. G. Hess ◽

P. A. Bachmann ◽

G. Baljer

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Intestinal Mucosa ◽

Enterotoxigenic Escherichia Coli ◽

Small Intestinal Mucosa ◽

E Coli ◽

Alkaline And Acid Phosphatase ◽

Tissue Compartments ◽

Increased Activity ◽

Dual Infections

The effect of rotavirus and enterotoxigenic Escherichia coli, administered in different sequences, on alkaline and acid phosphatase, leucinaminopeptidase, β-galactosidase, and succinicdehydrogenase of the intestinal mucosa of cesarian-derived, colostrum-deprived calves was investigated. Decrease in enzyme activity was most prominent in dual infections; it also occurred in parts of the small intestine in monoinfected animals. Increases in enzyme activity involved totally either one or all tissue compartments (crypt, basal villus area. villus tips). Increased activity was present in enteric mucosae that were either not affected or were only slightly affected by rotavirus or enterotoxigenic E. coli. We interpret the increase in enzyme activity as an adaptation of the enteric mucosa to maintain the absorptive function.

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