Ribosomal mutation in Escherichia coli affecting membrane stability

Summary Mutations in ribosomal protein L6 cause (i) loss of viability of cells at 0° C, which can be prevented by the presence of sodium chloride or 20% sucrose in the medium, (ii) influx of compounds at low temperature that normally cannot penetrate, and (iii) a defective assembly and maturation of 30S and 50S subunits at low temperature. It is proposed that abnormal interaction of immature subunits (or mutant 70S ribosomes) with the cytoplasmic membrane is responsible for triggering breakdown of membrane stability during cold shock.     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

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.     

Introduction of a Micrococcus plasmid in Escherichia coli

A 6-MDa plasmid (pMQV10), carrying cholesterol hydroxylase and streptomycin-resistance genes, from a gram-positive strain of Micrococcus sps., (RJ6) has been successfully transformed in gram-negative Escherichia coli K12 C600. pMQV10 is maintained stably and expresses its drug resistance in the new host.     

Escherichia coli and its chromosome

The Escherichia coli chromosome is a circular DNA molecule that is approximately 1000 times compacted in the living cell, where it occupies approximately 15% of the cellular volume. The genome is organized in a way that facilitates chromosome maintenance and processing. Despite huge efforts, until recently little has been known about how the chromosome is organized within cells, where replication takes place, and how DNA is segregated before cell division. New techniques for labeling genetic loci and molecular machines are allowing the simultaneous tracking of genetic loci and such machines in living cells over time. These studies reveal remarkable organization, yet a highly dynamic flux of genetic loci and macromolecules. It seems likely that the cellular positioning of chromosomal loci is the outcome of the formation of two chromosome arms (replichores) by replication, followed by sequential chromosome segregation, rather than from the presence of cellular positioning markers.     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

[目的]为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略.[方法]将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒.将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基...     

Molecular characterisation of the Stc mutation of Escherichia coli K-12

The previously described Stc ^- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF⁻ to OmpF⁺. Restriction mapping of chromosomal DNA from Stc ⁺ and Stc⁻ strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc⁻ phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc⁻ phenotype involves increase in ompF expression.     

2株可感染大肠埃希菌工程菌的噬菌体的鉴定与分析

大肠埃希菌来源的基因工程菌是应用最为频繁的工程菌,但在基因工程菌规模化制备生物活性制剂的过程中常常会被噬菌体感染。通过对鸡粪中噬菌体大量筛选及鉴定,对工程菌防御相应噬菌体感染机制开展基础研究。实验以大肠埃希菌工程菌为宿主菌（CICC编号：10424）,采用双层琼脂平板法从鸡场粪样中分离噬菌体,结果获得2株噬菌体,对其进行形态学鉴定。经透射电镜观察发现一株(CX)为短尾噬菌体,其头部外廓呈长六角形,非收缩性尾部,其噬菌斑清晰透亮,周围无晕环,裂解性较强;另一株(B1X)为长尾科噬菌体,其噬菌斑呈双层环状,中心澄清透明,直径约0.8~1.3 mm,外环呈半透明,云雾状区域,宽约0.8~1.3 mm。可进一步研究这2株噬菌体的侵染机制。     

Mutagenic DNA repair in Escherichia coli

Summary The photoreversibility of UV-induced mutations to Trp⁺ in strain Escherichia coli WP2 uvrA trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair.The results are discussed in the light of current models of UV mutagenesis.     

Analysis of rpsD mutations in Escherichia coli

Summary A certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

Ribosomal protein modification in Escherichia coli

Summary A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.     

Ribosomal protein modification in Escherichia coli

Summary Among mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.Previous paper in this series is Isono and Isono (1980)     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.