Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti.

Summary RP4-prime plasmids containing chromosomal fragments of either Escherichia coli or Rhizobium meliloti were constructed in vitro. When introduced into E. coli or R. meliloti respectively, they promoted a polarized transfer of the chromosome as demonstrated either by the gradient of transfer of various markers or by the study of the genetic constitution of recombinants. In E. coli, mobilization was shown to be dependent upon the presence of a functional rec A system. Inheritance of markers was due to their integration into the chromosome of the recipient as shown by the need for a functional rec A system in the recipient E. coli or by mobilization of recessive markers in R. meliloti. The system described could be applied to genetic mapping in any Gram negative bacteria.     

Site-specific properties of Tn7 transposition into the E. coli Chromosome

Summary A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.     

The sequence of IS4

Summary IS-elements are devoid of easily recognizable transacting functions and exert their visible effects in the position cis only (recent reviews Calos and Miller 1980; Starlinger 1980). It has been a matter of debate, whether these elements encode functions for their own transposition. In the case of the E. coli IS-elements this could not easily be determined by genetic methods, because most of these elements are present in several copies (Saedler and Heiss 1973; Deonier et al. 1979). In the case of the IS-elements flanking transposons, evidence has recently been brought forward that these carry the transposition specificity (Rothstein et al. 1980; Kleckner 1980; Grindley 1981).IS4 is present in one copy only in several E. coli K12 strains and should, therefore, be suitable for genetic and physiological studies (Chadwell et al. 1979). It has been cloned from several sites on the E. coli chromosome in pBR322 (Klaer and Starlinger 1980). Here we report the DNA sequence of IS4 which contains an open reading frame for 442 amino acids, and of the junctions of this element with surrounding DNA at three different sites in E. coli chromosome.     

Genetic Mapping of Cold Resistance Gene of Escherichia coli

Using cold resistant mutants, MET1 and MET2, obtained from Escherichia coli K-12, genetic mapping of the cold resistance gene(s) of E. coli was performed by the conjugation and transduction techniques. The gene(s) was confirmed to be located close to trpB at 28 min (revised chromosome linkage map, 1983) on the E. coli chromosome.     

A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome

Many Salmonella typhimurium genes are required for bacterial entry into host cells. P22 transduction analysis has localized several invasion loci near minute 59 on the S. typhimurium chromosome. To further characterize the 59–60 min chromosomal region, we determined the physical and genetic map of 85 kb of S. typhimurium DNA between srl and cysC. It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K-12. We examined whether other S. typhimurium genes on the 85 kb of DNA were similarly absent from E. coli We found that a contiguous 40 kb fragment of the S. typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E. coli K-12 chromosome and may represent a pathogenicity island. We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.     

Does experimental evolution reflect patterns in natural populations? E. coli strains from long-term studies compared with wild isolates

Our results show that experimental evolution mimics evolution in nature. In particular, only 1000 generations of periodic recombination with immigrant genotypes is enough for linkage disequilibrium values in experimental populations to change from a maximum linkage value to a value similar to the one observed in wild strains of E. coli. Our analysis suggests an analogy between the recombination experiment and the evolutionary history of E. coli; the E. coligenome is a patchwork of genes laterally inserted in a common backbone, and the experimental E. coli chromosome is a patchwork where some sites are highly prone to recombination and others are very clonal. In addition, we propose a population model for wild E. coli where gene flow (recombination and migration) are an important source of genetic variation, and where certain hosts act as selective sieves; i.e., the host digestive system allows only certain strains to adhere and prosper as resident strains generating a particular microbiota in each host. Therefore we suggest that the strains from a wide range of wild hosts from different regions of the world may present an ecotypic structure where adaptation to the host may play an important role in the population structure. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12

Background  

An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.     

A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...     

On the regulation of D-ribose metabolism in E. coli B-r. II. Chromosomal location and fine structure analysis of the D-ribose permease and D-ribokinase structural genes by P 1 transduction

Summary The genetic mapping and fine structure analysis of the d-ribose gene in Escherichia coli B/r has been studied. Findings indicate that the structural genes for the d-ribokinase and d-ribose permease map closely linked to the ara-leu region of the chromosome in contrast to their location in the isoleucine-valine region at 73.5 min in E. coli K12. Two polarity mutants, AB7 and AB36, were found to map at the left end of the d-ribokinase gene thus supporting the proposed d-ribokinase-d-ribose permease operon for the d-ribose catabolic enzymes in E. coli B/r.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The mechanisms driving bacterial chromosome segregation remain poorly characterized. While a number of factors influencing chromosome segregation have been described in recent years, none of them appeared to play an essential role in the process comparable to the eukaryotic centromere/spindle complex. The research community involved in bacterial chromosome was becoming familiar with the fact that bacteria have selected multiple redundant systems to ensure correct chromosome segregation. Over the past few years a new perspective came out that entropic forces generated by the confinement of the chromosome in the crowded nucleoid shell could be sufficient to segregate the chromosome. The segregating factors would only be required to create adequate conditions for entropy to do its job. In the article by Yazdi et al. ( 2012 ) in this issue of Molecular Microbiology, this model was challenged experimentally in live Escherichia coli cells. A Fis–GFP fusion was used to follow nucleoid choreography and analyse it from a polymer physics perspective. Their results suggest strongly that E. coli nucleoids behave as self‐adherent polymers. Such a structuring and the specific segregation patterns observed do not support an entropic like segregation model. Are we back to the pre‐entropic era?     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Chromosomal loci for 16S ribosomal RNA in Escherichia coli

Summary Genetic loci for 16S ribosomal RNA (rRNA) on the Escherichia coli chromosome were determined using the K-sequence, a characteristic oligonucleotide of strain K12, as a genetic marker. Oligonucleotide analyses of 16S rRNA from various recombinants between strain K12 and strain B(H) showed that the loci for 16S rRNA containing the K-sequence were near the metB locus which was at 77 min. on the chromosome map.     

Integration and Intramolecular Transposition of the TnBP3 Transposon ofBordetella pertussis in the Escherichia coliK12 Cells Mutant for the Hpr Protein of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussisinto the chromosome of Escherichia coliand transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsHdevoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metYgene sequence, which resulted in restoration of the Met⁺phenotype. The integration and transposition events were only observed in the E. colicells carrying the ptsH ⁺allele. The ptsHmutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsHmutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coliK12.     

Branched Escherichia coli cells

We report that the normally rod-shaped bacterium Escherichia coli can form branched cells. These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g. in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication. Often, chromosome DNA was found to be located in the branch point of the cells. The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased. The genetic background of the strains also affected the tendency to branch. Furthermore, electron microscopy of thin-sectioned branched cells revealed additional membrane-like structures, which were not observed in wild-type cells. Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E. coli are discussed.     

A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication     

Genetic Recombination in Klebsiella pneumoniae An Approach to Genetic Linkage Mapping

Genetic recombination was observed between two different strains of Klebsiella pneumoniae, which is a non-motile and encapsulated bacterium belonging to the family Enterobacteriaceae and has about 55% of its DNA content as GC. The mode of recombination seemed to be similar to that of the F-factor mediated conjugation in Escherichia coli. One strain acted as the donor and the other as the recipient, and a relatively large fragment of the donor's chromosome was transferred unilaterally and unidirectionally by cell to cell contact. No genetic factor which is associated with the recombination has been identified. The genetic linkage map of K. pneumoniae was analyzed various mutants derived from the two strains. It was found that the 28 markers so far investigated were arranged linearly in a single linkage group, and that the genetic linkage map of K. pneumoniae, like that of E. coli, could be considered circular. The proposed genetic linkage map of K. pneumoniae was quite similar to that of E. coli or Salmonella typhimurium. The close similarities in this map among the three species suggest a possibility that K. pneumoniae may have differentiated from an ancestor common all three species.     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

A role for topoisomerase III in Escherichia coli chromosome segregation

The cellular function of Escherichia coli topoisomerase III remains elusive. We show that rescue of temperature‐sensitive mutants in parE and parC (encoding the subunits of the chromosomal decatenase topoisomerase IV) at restrictive temperatures by high‐copy suppressors is strictly dependent on topB (encoding topoisomerase III). Double mutants of parEΔtopB and parCΔtopB were barely viable, grew slowly, and were defective in chromosome segregation at permissive temperatures. The topB mutant phenotype did not result from accumulation of toxic recombination intermediates, because it was not relieved by mutations in either recQ or recA. In addition, in an otherwise wild‐type genetic background, ΔtopB cells treated with the type II topoisomerase inhibitor novobiocin displayed aberrant chromosome segregation. This novobiocin sensitivity was attributable to an increased demand for topoisomerase IV and is unlikely to define a new role for topoisomerase III; therefore, these results suggest that topoisomerase III participates in orderly and efficient chromosome segregation in E. coli.