The effect of codon usage on the oligonucleotide composition of the E. coli genome and identification of over- and underrepresented sequences by Markov chain analysis.

As shown in the accompanying paper (5), the oligonucleotide composition of the E. coli genome is highly asymmetric for sequences up to 6 bp in length when ranked from highest to lowest abundance. We show here that this largely reflects codon usage because heavily used codons were found in the highly abundant oligomers whereas rarely used codons, with some exceptions, occurred in sequences in low abundance. Furthermore, linear regression analysis revealed a strong correlation between the frequencies of each trinucleotide and its usage as a codon. Dinucleotides are also not randomly distributed across each codon position and the dinucleotide composition of genes that are transcribed but not translated (rRNA and tRNA genes) was highly related to that seen in genes encoding polypeptides. However, 45 tetra-, 8 penta-, and 6 hexanucleotides were significantly over- or underabundant by Markov chain analysis and could not be accounted for by codon usage. Of these underrepresented sequences, many were palindromes, including the Dam methylation site.     

Escherichia coli with a linear genome

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.     

Dependence of Escherichia coli hyperbaric oxygen toxicity on the lipid acyl chain composition.

This study examines certain membrane-related aspects of oxygen poisoning in Escherichia coli K1060 (fabB fadE lacI) and its parent strain, K-12 Ymel. Cells were grown to exponential or stationary phase in a minimal medium and exposed to air plus 300 lb/in2 of O2 as a suspension in minimal salts. After an initial lag, both strains lost viability with apparent first-order kinetics. Hypebaric oxygen was more toxic to cells harvested during the exponential phase of growth than to cells harvested from the stationary phase of growth for both strains K-12 Ymel and K1060. Control suspensions exposed to air plus 300 lb/in2 of N2 did not lose viability during a 96-h exposure. The sensitivity of the unsaturated fatty acid auxotroph, strain K1060, to hyperbaric oxygen increased as the degree of unsaturation of the fatty acid supplement increased. Cells grown with a cyclopropane fatty acid (9,10=methylenoctadecanoate) were the most resistant; cells grown with a monounsaturated fatty acid (oleate) were intermediate; and those grown with polyunsaturated fatty acids (linoleate and linolenate) were most sensitive to hyperbaric oxygen. The parent strain, K-12 Ymel, lost viability in hyperbaric oxygen most similarly to strain K1060 supplemented with oleate. To determine the relative effect of hyperbaric oxygen on the survival of E. coli with saturated membranes, substrains of K1060 were selected for growth on 12-methyltetrade-canoate or on 9 or 10-monobromostearate. Substrains grown with a saturated fatty acid supplement were equally or more sensitive to hyperbaric oxygen than when the same substrains were grown with a cyclopropane fatty acid supplement. The lipid acyl chain composition was determined in E. coli K1060 before and after exposure to hyperbaric oxygen or hyperbaric nitrogen. The proportion of nonsaturated acyl chain lipid of either the oleate- or the 9,10-methyleneoctade-canoate-supplemented K1060 remained unchanged after hyperbaric gas exposure. In strain K1060 supplemented with linoleate and grown to stationary phase, however, the relative unsaturated acyl chain content after hyperbaric exposure decreased in both gases. This finding prompted an investigation of the role of lipid oxidation in hyperbaric oxygen toxicity. Assays of potential lipid oxidation products were performed with linoleate-grown cells. The lipid hydroperoxide and peroxide content of the lipid extract increased by 6.9 times after 48 h of air plus 300 lb/in2 of O2; malondialdehyde and fluorescent complex lipid oxidation products showed much smaller or no changes. Lipid extracts from hyperbaric oxygen-exposed cells were not toxic to viable E. coli K1060, nor did they increase the rate of loss of viability in cells simultaneously exposed to hyperbaric oxygen. Linoleic acid hydroperoxide at 1.0 mM had no effect on the viability of E. coli K-12 Ymel and only marginally decreased the viability of E. coli K1060 supplemented with linoleate. We conclude that the kinetics of oxygen toxicity in E...     

Functional insights from structural predictions: analysis of the Escherichia coli genome

Fold assignments for proteins from the Escherichia coli genome are carried out using BASIC, a profile-profile alignment algorithm, recently tested on fold recognition benchmarks and on the Mycoplasma genitalium genome and PSI BLAST, the newest generation of the de facto standard in homology search algorithms. The fold assignments are followed by automated modeling and the resulting three-dimensional models are analyzed for possible function prediction. Close to 30% of the proteins encoded in the E. coli genome can be recognized as homologous to a protein family with known structure. Most of these homologies (23% of the entire genome) can be recognized both by PSI BLAST and BASIC algorithms, but the latter recognizes an additional 260 homologies. Previous estimates suggested that only 10-15% of E. coli proteins can be characterized this way. This dramatic increase in the number of recognized homologies between E. coli proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms. Knowing protein structure adds a new dimension to our understanding of its function and the predictions presented here can be used to predict function for uncharacterized proteins. Several examples, analyzed in more detail in this paper, include the DPS protein protecting DNA from oxidative damage (predicted to be homologous to ferritin with iron ion acting as a reducing agent) and the ahpC/tsa family of proteins, which provides resistance to various oxidating agents (predicted to be homologous to glutathione peroxidase).     

Dynamic flexibility in the Escherichia coli genome.

Empirical rules based on tetranucleotide parameters were presented to predict the structural parameters twist (Omega), roll (rho), tilt (tau) and slide (D(y)). A statistical mechanical model was used to analyze the flexibility of the Escherichia coli genome. The replication terminus region displayed a low level of flexibility. A strong correlation can be seen between G+C content and flexibility. Average flexibilities in the coding regions were found to be significantly larger than those in non-coding regions. The flexible characteristics in the 5'-neighborhood of the coding regions and in three class sigma promoter sequences in the E. coli genome were also analyzed.     

Organization of the Escherichia coli genome

A review of literature data reveals that for the last years, the molecular biology techniques have been of an increasing use in the study of the Escherichia coli genome, having supplemented the standard genetic mapping. For the proper understanding of the Escherichia coli genome organization, recombinational events occurring in the course of evolution should be considered. The bacterial genome seems to carry traces of both "long-term" evolution, possibly responsible for appearance of the bacterial cell itself, and "current" evolution, consisting mainly of periodic genome entering by new plasmid-originated genes. It is supposed that in the process of stabilization within a genome, every new gene undergoes a stage of the "transgene", that is the gene situated in a transposon on the chromosome. In parallel with integration of new genes into the genome, some genes deleting should also take place. The formation of deletions could occur by unequal crossing over in segments of direct homologous repeats which seem to be ordinarily revealed in the experimental study of the tandem gene duplications.     

Alteration of the phospholipid composition of Escherichia coli through genetic manipulation

In order to study the function of individual phospholipids, we have constructed a strain of Escherichia coli in which the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin can be regulated. In this strain (HDL1001) the normal expression of the phosphatidylglycerophosphate synthase does not occur due to the presence of the pgsA30 allele (Heacock, P. N., and Dowhan, W. (1987) J. Biol. Chem. 262, 13044-13049). A second chromosomal copy of the pgsA gene is fused to the lacOP region in single copy within the lac operon. Strain HDL1001 is absolutely dependent for growth on an inducer of the lac operon. In addition, the level of the pgsA gene product, the content of the two major acidic phospholipids, and the growth rate are dependent on the level of inducer in the growth medium. Cells remain viable in the absence of inducer as evidenced by a rapid return to normal growth after the readdition of inducer. The growth rate and phospholipid composition are affected only after the level of phosphatidylglycerophosphate synthase drops below about 15% of normal levels; both phosphatidic acid and (d)CDP-diacylglycerol also begin to increase to significant levels. At the point of cell arrest the level of the major acidic phospholipids is reduced by about 90% of wild type levels.     

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.     

大肠杆菌最小基因组分析和删减进展

大肠杆菌是基础研究最透彻、应用广泛的微生物,构建含减小甚至是最小基因组的大肠杆菌将为合成生物学的研究和应用提供理想的底盘生物。介绍了大肠杆菌最小基因组的生长与繁殖必需基因的生物信息学分析和实验鉴定,基因组敲除技术,以及删减基因组的大肠杆菌菌株的构建和应用等方面的研究进展。     

In situ analysis of the higher-order genome structure in a single Escherichia coli cell

We developed a novel method for the in situ analysis of the higher-order structure of an individual genome from a single Escherichia coli cell using laser tweezers. Initially, condensed DNA was stably grasped by a laser without any chemical modification and without physical attachment to an artificial object such as micro-plastic beads. Under optical transport, the trapped genome gradually unfolded in solution due to viscous friction. Interestingly, the nucleoid DNA from a log-phase cell is almost fully elongated, whereas in the stationary phase, unfolding of the nucleoid is characterized by step-wise elongation of 1.7-5.1 microm, corresponding to a size of 5-15 kbp, and a few tightly packed domains remain along the DNA chain. This suggests the coexistence of tightly packed and swollen domains in the genome in the stationary phase.     

Systematic mutagenesis of the Escherichia coli genome

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the lambdaRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.     

METALGEN.DB: metabolism linked to the genome of Escherichia coli, a graphics-oriented database

The table of ‘biochemical pathways’ published byBoehringer is considered as the most exhaustive document dealingwith intermediary metabolism. The present work consisted ofconstructing a database in which is collected everything knownto date about intermediary metabolism: metabolic path bioreactions,metabolites, enzymatic activities, genes and regulation. Dataon genes make it possible to establish a link with existingcomputer structures specialized for genomic sequences. The presentstructure of the database presented here consists of three environments.The first environ is graphic; it is modeled upon the Boehringertable, but is more convenient to use. The second environmentprovides the user with all kinds of manipulations of data (entries,corrections, deletions, consistency, limiting redundancy, etc.).These operations either are automatic or depend on a user-friendlyinterface (menus selected with the mouse) and so allow biologistsat all levels to use the database and to develop it for theirown particular needs. Finally, the third environment offersrious procedures that make it possible to carry out multicriteriaresearch on all the data in the database at once. This databasecan thus be substituted for the Boehringer table as a standardreference. It is also a tool that can be used to develop informationsystems not only in the domain of metabolism but also in thestudy of genomes (modeling, analysis of data, predictions, etc.).     

EcoGene: a genome sequence database for Escherichia coli K-12

The EcoGene database provides a set of gene and protein sequences derived from the genome sequence of Escherichia coli K-12. EcoGene is a source of re-annotated sequences for the SWISS-PROT and Colibri databases. EcoGene is used for genetic and physical map compilations in collaboration with the Coli Genetic Stock Center. The EcoGene12 release includes 4293 genes. EcoGene12 differs from the GenBank annotation of the complete genome sequence in several ways, including (i) the revision of 706 predicted or confirmed gene start sites, (ii) the correction or hypothetical reconstruction of 61 frame-shifts caused by either sequence error or mutation, (iii) the reconstruction of 14 protein sequences interrupted by the insertion of IS elements, and (iv) pre-dictions that 92 genes are partially deleted gene fragments. A literature survey identified 717 proteins whose N-terminal amino acids have been verified by sequencing. 12 446 cross-references to 6835 literature citations and s are provided. EcoGene is accessible at a new website: http://bmb.med.miami.edu/EcoGene/EcoWeb. Users can search and retrieve individual EcoGene GenePages or they can download large datasets for incorporation into database management systems, facilitating various genome-scale computational and functional analyses.