Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations (∼ 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrück and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although ∼ 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had ∼ 4039 coding sequences occupying ∼ 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was identified. Complex deletions (dense clusters of small deletions and substitutions) apparently removed nonessential genes from ∼ 30 sites in the basic genomes.     

The origin of phage virology.

The history of bacteriophage (phage) had its start in 1915, when Twort isolated an unusual filterable and infectious agent from excrete of patients struck by diarrhoea; this discovery was followed by an analogous, and probably independent, finding of d'Hérelle in 1917. For several years phage research made scant progress but great attention was paid to the question of phage nature, which saw the contrast between d'Hérelle and Bordet's views (living against chemical nature, respectively). This situation changed with the independent discovery of lysogeny, in 1925, thanks to Bordet and Bail: this phenomenon was considered of genetical origin, a view that Wollman interpreted by assimilating the properties of phage to those of gene (according to a previous idea of Muller). In the 1930s, Burnet's work opened a new era by demonstrating the occurrence of several species of phages and their antigenic property. In the same period, the physical and chemical characteristics of these viruses were disclosed thanks, in particular, to the work of Schlesinger, who first demonstrated that a virus (phage) was constituted of nucleoproteins. The peculiarity of phage was finally shown after the invention of electron microscope: H. Ruska, in 1940, and Anderson and Luria in the next years, obtained the first images of tailed phages, a finding that strongly helped the investigation on the first steps of the infection process. The decisive impulse to phage virology came from Delbrück, a physicist who entered biology giving it a new arrangement. The so-called "phage group" assembled brilliant minds (Luria, Hershey and Delbrück himself, and later a dozen of other scientists): this group faced three fundamental questions of phage virology, i.e., the mechanisms of attack, multiplication and lysis. In ten years' time, phage virology became an integrant part of molecular biology, also thanks to the discovery of the genetical properties of DNA: in such scientific context, Delbrück, Luria and Hershey's works emerged for the absolute excellence of their results, which led such scientists to Nobel prize. Lysogeny was however neglected by the phage group: this singular property shared by bacteria and phages was instead investigated by Lwoff's group, in Paris, and explained in its fundamental features during the 1950s. The "phage's saga" has gone on being an important division of molecular biology till today, and its history is far from being over.     

Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the λ attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have > 99% sequence identity over ∼ 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.     

Proposal of Corynebacterium propinquum sp. nov. for Corynebacterium group ANF-3 strains

Abstract Chemotaxonomic and genomic studies were performed on seven Corynebacterium group ANF-3 strains isolated from human sources. All these strains possess cell wall component type IV ( meso -diaminopimelic acid, arabinose and galactose), corynemycolic acids and a G+C content of DNA of 57 to 59 mol%. These results confirm that they can be placed in the genus Corynebacterium . Six of these strains were found to constitute a tight hybridization group distinct from named Corynebacterium species or related organisms. This genomic group constitutes a new species which can be identified within the genus Corynebacterium by ribotyping or phenotypic tests and for which the name Corynebacterium propinquum is proposed. The type strain is strain B 77159 (= Collection of the Institut Pasteur CIP 103792).     

Estimating the transfer rates of bacterial plasmids with an adapted Luria–Delbrück fluctuation analysis

To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need reliable estimates of their rate of transfer between bacterial cells. Current assays to measure transfer rate are based on deterministic modeling frameworks. However, some cell numbers in these assays can be very small, making estimates that rely on these numbers prone to noise. Here, we take a different approach to estimate plasmid transfer rate, which explicitly embraces this noise. Inspired by the classic fluctuation analysis of Luria and Delbrück, our method is grounded in a stochastic modeling framework. In addition to capturing the random nature of plasmid conjugation, our new methodology, the Luria–Delbrück method (“LDM”), can be used on a diverse set of bacterial systems, including cases for which current approaches are inaccurate. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. In contrast, the LDM overcomes obstacles of traditional methods by avoiding restrictive assumptions about growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude.

Plasmid transfer can often spread resistance between important clinical pathogens. This study shows that widely used methods can lead to biased estimates of plasmid transfer rate by several orders of magnitude, and presents a new approach, inspired by the classic Luria-Delbrück approach, for accurately assessing this fundamental rate parameter     

X-Ray and Ultraviolet Sensitivity of Synchronously Dividing Escherichia coli

A paper pile filtration technique was used to obtain synchronously dividing populations of E. coli strains B and B/r from cultures in the exponential growth phase. Three generations of highly phased cell division were obtained by rapid pressure filtration which selected approximately 1 per cent of the exponentially growing culture. The sensitivity of E. coli strain B to x-ray and UV inactivation as a function of the cell division cycle was determined on synchronous populations. E. coli strain B showed a sharp decrease in sensitivity to inactivation by both radiations in the middle of the division cycle, and a further decrease near the end of the cycle. The sensitivity of E. coli strain B/r to x-irradiation was also investigated. Only the mid-cycle decrease in sensitivity was found during the division cycle of this strain. It was concluded that the repetition of the observed sensitivity patterns in both strains through the first three cycles after synchronization indicates that the same basic sensitivity patterns are probably also present in the individual cells of an exponential phase culture.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.     

Discussion on research methods of bacterial resistant mutation mechanisms under selective culture—uncertainty analysis of data from the Luria-Delbrück fluctuation experiment

Whether bacterial drug-resistance is drug-induced or results from rapid propagation of random spontaneous mutations in the flora prior to exposure, remains a long-term key issue concerned and debated in both genetics and medicinal fields. In a pioneering study, Luria and Delbrück exposed E. coli to T1 phage, to investigate whether the number of resistant colonies followed the Poisson distribution. They deduced that the development of resistant colonies is independent of phage presence. Similar results have since been obtained on solid medium containing antibacterial agents. Luria and Delbrück??s conclusions were long considered a gold standard for analyzing drug resistance mutations. More recently, the concept of adaptive mutation has triggered controversy over this approach. Microbiological observation shows that, following exposure to drugs of various concentrations, drug-resistant cells emerge and multiply depending on the time course, and show a process function, inconsistent with the definition of Poisson distribution (which assumes not only that resistance is independent of drug quantity but follows no specific time course). At the same time, since cells tend to aggregate after division rather than separating, colonies growing on drug plates arise from the multiplication of resistant bacteria cells of various initial population sizes. Thus, statistical analysis based on equivalence of initial populations will yield erroneous results. In this paper, 310 data from the Luria-Delbrück fluctuation experiment were reanalyzed from this perspective. In most cases, a high-end abnormal value, resulting from the non-synchronous variation of the two above-mentioned time variables, was observed. Therefore, the mean value cannot be regarded as an unbiased expectation estimate. The ratio between mean value and variance was similarly incomparable, because two different sampling methods were used. In fact, the Luria-Delbrück data appear to follow an aggregated, rather than Poisson distribution. In summary, the statistical analysis of Luria and Delbrück is insufficient to describe rules of resistant mutant development and multiplication. Correction of this historical misunderstanding will enable new insight into bacterial resistance mechanisms.     

Inhibition of Escherichia coli B by homoarginine

Homoarginine inhibits the growth of Escherichia coli B, but not of E. coli K-12. These two strains also differ in regard to repressibility of the arginine-forming enzymes. In K-12, arginine acts as a repressor whereas in B it does not. The latter difference is determined by different alleles of a regulator gene, arg R. In K-12 × B crosses, it was shown that the genetic determinant for homoarginine sensitivity is closely linked to or identical with arg R. Homoarginine-resistant mutants of B were isolated. The biochemical mechanism of homoarginine inhibition is not known. However, whether or not a strain is sensitive to homoarginine seems to depend on the intracellular level of arginine. In B this level is relatively low and inflexible as a result of the action of a repressor whose formation is determined by the B-specific allele of arg R.     

Transductional construction of aspartase-hyperproducing strain of Escherichia coli B

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile⁻ strain derived from strain EAPc7 using the Ile⁺ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA⁺ recombinant plasmid pYT471 (pBR322-aspA⁺), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA⁺-recombinant plasmid pYT471.     

DNA synthesis in pretreated and ultraviolet-irradiated cells ofEscherichia coli B/rHcr + andHcr −

DNA synthesis after the ultraviolet irradiation was followed in the excision proficient strainEscherichia coli B/rHcr ⁺, in which the ability to excise thymin dimers was suppressed by a preirradiation inhibition of DNA and protein syntheses and in the excision deficient strainEscherichia coli B/rHcr ^?. Synthesis of pulse-labeled DNA, its stability and semiconservative DNA synthesis were compared in both strains. It was found that cells of theHcr ⁺ strain restore semiconservative DNA synthesis and the pulselabeled DNA appears stable, in spite of the fact that dimers are not excised under these conditions. On the other hand, cells of theHcr ^? strain are unable to restore semiconservative DNA synthesis and the pulselabeled DNA is degraded. As the repair by the excision of dimers under the used experimental conditions may be excluded in both strains, it is possible to assume that activity of enzymes included in theHcr ⁺ marker is prerequisite for restoring the DNA synthesizing system in theHcr ⁺ strain.     

Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against Mycobacterium bovis Challenge

Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.     

Amino Acid Differences in a 30S Ribosomal Protein from Two Strains of Escherichia coli

The 30S ribosomal proteins of the K-12 and B strains of Escherichia coli differ in at least one protein component. This component, which is allelic in the two strains, has been isolated from both organisms. Amino acid analyses show that the protein from strain B contains between 20 and 28 more amino acids than does the analogue protein from strain K-12.     

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.     

The Streptomycin-Sulfadiazine-Tetracycline Antimicrobial Resistance Element of Calf-Adapted Escherichia coli Is Widely Distributed among Isolates from Washington State Cattle

Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.     

Energy metabolism of Bdellovibrio bacteriovorus

The P/O ratio of Bdellovibrio bacteriovorus, strain Bd 109 Sa, was evaluated by two different methods based on the determination of energy-rich phosphate bonds and either NADH oxidation or oxygen-uptake. P/O values calculated on the basis of NADH oxidation were up to 6, which has to be regarded as being overestimated. P/O values calculated from energy-rich phosphate bonds and oxygen uptake were around 2. The P/O values determined for Escherichia coli B were similar. The loss of phosphorylation efficiency at one site is discussed.The ATP pool turnover rate of Bdellovibrio was 8/min during endogenous respiration and 24/min during substrate respiration. The corresponding values in Escherichia coli B were 3/min and 38/min.This study was performed at the University of Hamburg (Institut für Allgemeine Botanik, Abteilung Mikrobiologic).     

Hyperproduction of aspartase by a catabolite repression-resistant mutant of Escherichia coli B harboring multicopy aspA and par recombinant plasmids

Recombinant plasmids bearing the Escherichia coli K-12 aspartase gene (aspA) and the plasmid partition locus (par) were introduced into a catabolite repression-resistant strain of E. coli B, AT202, constructed by mutational and transductional methods. Plasmid pNK101(pBR322-aspA-par) was stably maintained in cells of AT202 even after 30 cell generations, while pYT471(pBR322-aspA), which bore no par locus, was lost at high frequencies from the host cells. Strain AT202 harboring pNK101 produced 3-fold and 80-fold more aspartase than the wild-type E. coli B harboring pNK101 and the wild-type E. coli B strain, respectively. The maximum amount of aspA product (aspartase) was 40–45% of the total cellular protein.     

Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation in Sphingobium indicum B90A

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform β- and δ-hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with β- and δ-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including β-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade β- and δ-HCH.     

Selection against hypermutability in Escherichia coli during long term evolution

Summary A population of a mutT strain of E. coli was maintained in a chemostat for 2,200 generations. Afterwards the rate, of mutation to resistance to three antibiotics was determined by the Luria-Delbrück fluctuation test. It was found that the strain had a distinctly reduced mutability after the long-term cultivation compared with the original strain. Nevertheless the mutability was still much higher than that of a wild-type strain. After transduction of the mutT gene into another genetic backgroud the transductants showed the same mutability as the original strain indicating that the mutT allele itself had not changed. Our results support the hypothesis that under new environmental conditions mutator strains have an advantage due to their more efficient production of beneficial mutations. After optimal adaptation there is selection against high mutation rates due to the increased mutational load in the mutator population.