Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

Effect of Mutations for the ruvABC Genes on Recombination between Direct DNA Repeats in Escherichia coli Strains Carrying Extended Tandem Duplications

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants forruvABCgenes. Homologous recombination in duplications of rec ⁺ strains and in recBC sbcB, recQand recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.     

Chromosomal mobilization from a recA mutant of Pseudomonas putida.

Summary A methyl methane sulfonate sensitive mutant of P. putida strain PpG1 is also extremely sensitive to UV-rays, compared to parent wild type cells. This mutant behaves typically as recombination less (recA) mutants of Escherichia coli and Pseudomonas aeruginosa, since as a recipient, it exhibits extremely low frequency of recombination following conjugational, transductional, and transformational gene transfer. Sex factor plasmids such as K-XYL or TOL can mobilize chromosomal genes equally well both from recA ⁺ and recA801 donor cells, suggesting that host recombination functions are not necessary for mobilization of chromosomal genes by such plasmids.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

A molecular model for conjugational recombination in Escherichia coli K12

Summary Conjugational recombination in Escherichia coli was investigated by measuring lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.     

On the ability of Salmonella typhimurium cells to form deoxycytidine nucleotides.

Summary The fate of the donor DNA after conjugation in Escherichia coli was studied through crosses with a Hfr lacZ5 donor and several F^- lacZ22 recipients. The fate of the donor allele was studied by assaying the -galactosidase activity formed by complementation between the lacZ5 allele and the lacZ22 allele. We used continuous cultures of the recipient in order to be able to study the fate of the donor DNA during many generations under constant physiological conditions. We could show that the donor DNA allele is inactivated in Rec⁺, recA171 and recB21 recipient cells. The inactivation rate depends on the nature of the recipient, Rec⁺ or recombination deficient, and especially in the case of the recombination deficient mutants on the growth rate of the recipient.     

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: Selection of mutants defective in photosynthesis

Summary Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Km^r) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Km^r gene into an AvaII restriction site of the gene psbD-1. All other harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.     

Instability of inhibited replication forks in E. coli

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication. A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.     

Mutants of Rhodopseudomonas sphaeroides useful in genetic analysis

Two kinds of mutants of Rhodopseudomonas sphaeroides that should be useful in extending genetic analysis of this organism have been isolated. One is deficient in recombination and has been used to isolate derivatives of the plasmid R 68.45 which incorporate chromosomal genes of R. sphaeroides. The other is apparently defective in a DNA restriction enzyme; transfer of plasmid borne chromosomal genes of R. sphaeroides from Escherichia coli back to R. sphaeroides is greatly enhanced in these mutants.In memory of R. Y. Stanier     

Complementation of Bacteriophage Induction and Recombination Defects in <Emphasis Type="Italic">Escherichia coli</Emphasis> RecA<Superscript>−</Superscript> Mutants by Expression of the Cloned T4 Bacteriophage <Emphasis Type="Italic">uvsX</Emphasis> Gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA⁻ mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present. Received: 11 February 2002 / Accepted: 12 April 2002     

The fertility of a smooth strain ofEscherichia coli

Auxotrophic mutants of the potentially pathogenic smooth strain ofEscherichia coli O55: B5 were isolated and used as recipients in mating experiments withEscherichia coli K12. The recombination frequency obtained in mating K12HfrH donor with O55 recipient was about 100 to 1,000 times lower than in K12×K12 cross. The F-prime donor O55 strain when backerossed with the K12 recipient again mated with low efficiency. Crosses with derivatives of O55 which were presumed to carry K12 host specificity genes were inconclusive and we cannot exclude the possibility that O55 restricts K12 DNA. A polyauxotrophic mutant of O55 with better mating ability was isolated. The improved genetic homology in K12 and O55 chromosomes could account for increased frequency of recombinants obtained. This strain was used to establish the location of the loci for O55 and B5 antigens. No close linkage was found between these two antigenic determinants, locus for O55 antigen being located close tohis marker and locus for B5 antigen rather totrp marker.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Mitochondrial genetics

Summary Cytoplasmic petite mutants of Saccharomyces cerevisiae carrying the gene conferring the resistance to chloramphenicol on one hand and the gene conferring the resistance to erythromycin on the other, have been crossed with each other. The two types of petites differed in the buoyant densities of their mitochondrial DNA. A novel type of evidence has been adduced, that the two genes are indeed located on mitochondrial DNA. Diploid petite recombinants were found, carrying both genes and containing not a mixture of the two parental DNAs but a new species of mitochondrial DNA of intermediate buoyant density. Recombination of mitochondrial genes involves therefore breakage and reunion of DNA molecules. New suppressiveness, different from the two parental ones, can result from the recombination of mitochondrial DNA. Recombination between petite mutants implies that the mitochondrial recombination enzymes have to be synthesized on cytosol ribosomes.     

Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12

Summary Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str ⁺ operon (Sm^s), has been modified in vitro by either irradiation with UV light, or by reaction with (±) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Amp^r transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination.     

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.     

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli

Summary Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F7₂ and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

The intrinsically disordered linker of E. coli SSB is critical for the release from single‐stranded DNA

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.