scholarly journals Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

2021 ◽  
Author(s):  
Shan Huang ◽  
Kaihang Wang ◽  
Stephen L. Mayo
Keyword(s):  
Escherichia Coli ◽  
Dna Cleavage ◽  
Genetic Manipulation ◽  
Direct Repeat ◽  
Clostridium Butyricum ◽  
Argonaute Protein ◽  
E Coli ◽  
Repeat Sequences ◽  
Chromosome Recombination ◽  
Chromosomal Recombination

Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

scholarly journals afa-8 Gene Cluster Is Carried by a Pathogenicity Island Inserted into the tRNAPhe of Human and Bovine Pathogenic Escherichia coli Isolates

2001 ◽  
Vol 69 (2) ◽  
pp. 937-948 ◽  
Author(s):  
Lila Lalioui ◽  
Chantal Le Bouguénec
Keyword(s):  
Escherichia Coli ◽  
Direct Repeat ◽  
Pathogenicity Island ◽  
Genomic Region ◽  
Open Reading Frames ◽  
Blood Isolate ◽  
Distinctive Features ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
K 12

ABSTRACT We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the afa-8 operon, encoding AfaE-VIII adhesin, from the human blood isolate Escherichia coli AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (pheR), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the afa-8 operon (46.4%) is lower than that of the E. coli K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI IAL862, we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenicafa-8-positive E. coli strains isolated from humans and animals, 25% of human pathogenic afa-8-negativeE. coli strains, and only 8% of fecal strains (P = 0.05), indicating that these sequences are strongly associated with the afa-8 operon and that this genetic association may define a PAI widely distributed among human and animal afa-8-positive strains. One of the distinctive features of this study is that E. coli AL862 also carries another afa-8-containing PAI (PAI IIAL862), which appeared to be similar in size and genetic organization to PAI IAL862 and was inserted into the pheV gene. We investigated the insertion sites of afa-8-containing PAI in human and bovine pathogenic E. coli strains and found that this PAI preferentially inserted into the pheV gene.

scholarly journals FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

2021 ◽  
Author(s):  
Zhixin Lyu ◽  
Atsushi Yahashiri ◽  
Xinxing Yang ◽  
Joshua W McCausland ◽  
Gabriela M Kaus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Single Molecule ◽  
Spatial Organization ◽  
Genetic Manipulation ◽  
Cell Wall Synthesis ◽  
Single Population ◽  
E Coli ◽  
Ftsz Ring ◽  
Synthesis Activity

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of ~ 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the ~ 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZ's treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

scholarly journals Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

1999 ◽  
Vol 181 (18) ◽  
pp. 5783-5789 ◽  
Author(s):  
M. L. Pato ◽  
M. Banerjee
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Bacteriophage Mu ◽  
Chromosomal Dna ◽  
E Coli ◽  
Repeat Sequences ◽  
Central Fragment ◽  
Entire Chromosome ◽  
Nucleoid Structure ◽  
Replicative Transposition

ABSTRACT The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.

scholarly journals Effect of Host Species on RecG Phenotypes in Helicobacter pylori and Escherichia coli

2004 ◽  
Vol 186 (22) ◽  
pp. 7704-7713 ◽  
Author(s):  
Josephine Kang ◽  
Don Tavakoli ◽  
Ariane Tschumi ◽  
Rahul A. Aras ◽  
Martin J. Blaser
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Replication Forks ◽  
E Coli ◽  
Repeat Sequences ◽  
Dna Repeat ◽  
H Pylori ◽  
Dna Repeat Sequences ◽  
Prokaryotic Evolution ◽  
Fundamental Mechanism

ABSTRACT Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.

scholarly journals Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

2006 ◽  
Vol 75 (4) ◽  
pp. 1661-1666 ◽  
Author(s):  
Theresa Deland Ho ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Enterohemorrhagic Escherichia Coli ◽  
Deletion Strain ◽  
Growth Defect ◽  
Intestinal Colonization ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

scholarly journals Fluoroquinolone action in mycobacteria: similarity with effects in Escherichia coli and detection by cell lysate viscosity.

1996 ◽  
Vol 40 (7) ◽  
pp. 1594-1599 ◽  
Author(s):  
K Drlica ◽  
C Xu ◽  
J Y Wang ◽  
R M Burger ◽  
M Malik
Keyword(s):  
Escherichia Coli ◽  
Dna Cleavage ◽  
Bactericidal Effect ◽  
Multidrug Resistant ◽  
E Coli ◽  
Cell Lysate ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
High Concentrations ◽  
Inhibitor Of Protein Synthesis

Fluoroquinolones are potent antibacterial agents that are being used as therapeutic agents for the treatment of multidrug-resistant tuberculosis. To better understand fluoroquinolone action in mycobacteria, the effects of ciprofloxacin were examined. DNA synthesis was inhibited rapidly in Mycobacterium smegmatis, DNA cleavage was readily observed by an empirical assay of cell lysate viscosity, and cell growth was blocked. These data are explained by the formation of gyrase-DNA-ciprofloxacin complexes that block replication fork movement. The bactericidal action of ciprofloxacin against M. smegmatis, Mycobacterium bovis BCG, and Escherichia coli occurred more slowly in cells with longer doubling times. The bactericidal effect against M. bovis BCG was partially blocked by pretreatment with chloramphenicol, an inhibitor of protein synthesis, and by very high concentrations of ciprofloxacin itself. Similar responses occur when E. coli is treated with ciprofloxacin. These similarities between E. coli and mycobacteria indicate that results from extensive fluoroquinolone studies with E. coli can be applied to mycobacteria. A simple viscometric assay of DNA cleavage is described. The assay is expected to be useful for screening new fluoroquinolone derivatives for increased effectiveness against clinically important bacteria.

scholarly journals Modulation of Gyrase-Mediated DNA Cleavage and Cell Killing by ATP

1998 ◽  
Vol 42 (5) ◽  
pp. 1022-1027 ◽  
Author(s):  
Tsai-Kun Li ◽  
Leroy F. Liu
Keyword(s):  
Escherichia Coli ◽  
Dna Cleavage ◽  
Important Determinant ◽  
Dna Gyrase ◽  
Bactericidal Action ◽  
Atpase Subunit ◽  
Fluoroacetic Acid ◽  
E Coli ◽  
Quinolone Antibiotics ◽  
Atp Pool

ABSTRACT An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and an aconitase inhibitor, fluoroacetic acid, both of which are known to lower the cellular ATP pool, protected Escherichia colicells from the bactericidal actions of gyrase poisons including quinolone antibiotics, nalidixic acid and ciprofloxacin, and the epipodophyllotoxins VP-16 and VM-26. Using purified E. coliDNA gyrase, we examined the effect of ATP on gyrase-mediated DNA cleavage in the presence of these gyrase poisons. ATP was shown to stimulate gyrase-mediated DNA cleavage from 10- to more than 100-fold in the presence of these gyrase poisons. ADP antagonized the stimulatory effect of ATP. Consequently, gyrase-mediated DNA cleavage induced by gyrase poisons is modulated by the ATP concentration/ADP concentration ([ATP]/[ADP]) ratio. Coumermycin A1, an inhibitor of the ATPase subunit of DNA gyrase, like ADP, also effectively antagonized the stimulatory effect of ATP on gyrase-mediated DNA cleavage induced by gyrase poisons. Furthermore, coumermycin A1, like DNP and fluoroacetic acid, also protected cells from the bactericidal action of gyrase poisons. In the aggregate, our results are consistent with the notion that the [ATP]/[ADP] ratio, through its modulatory effect on the gyrase-mediated DNA cleavage, is an important determinant of cellular susceptibility to gyrase poisons.

Chromosomal model for analysis of a long CTG/CAG tract stability in wild-type Escherichia coli and its nucleotide excision repair mutants

2007 ◽  
Vol 53 (7) ◽  
pp. 860-868 ◽  
Author(s):  
Sylwia T. Szwarocka ◽  
Paweł Stączek ◽  
Paweł Parniewski
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Excision Repair ◽  
Trinucleotide Repeat ◽  
Excision Repair ◽  
Repetitive Sequences ◽  
Lambda Phage ◽  
E Coli ◽  
Repeat Sequences ◽  
Nucleotide Excision ◽  
The Stability

Many human hereditary neurological diseases, including fragile X syndrome, myotonic dystrophy, and Friedreich’s ataxia, are associated with expansions of the triplet repeat sequences (TRS) (CGG/CCG, CTG/CAG, and GAA/TTC) within or near specific genes. Mechanisms that mediate mutations of TRS include DNA replication, repair, and gene conversion and (or) recombination. The involvement of the repair systems in TRS instability was investigated in Escherichia coli on plasmid models, and the results showed that the deficiency of some nucleotide excision repair (NER) functions dramatically affects the stability of long CTG inserts. In such models in which there are tens or hundreds of plasmid molecules in each bacterial cell, repetitive sequences may interact between themselves and according to a recombination hypothesis, which may lead to expansions and deletions within such repeated tracts. Since one cannot control interaction between plasmids, it is also sometimes difficult to give precise interpretation of the results. Therefore, using modified lambda phage (λInCh), we have constructed a chromosomal model to study the instability of trinucleotide repeat sequences in E. coli. We have shown that the stability of (CTG/CAG)68 tracts in the bacterial chromosome is influenced by mutations in NER genes in E. coli. The absence of the uvrC or uvrD gene products greatly enhances the instability of the TRS in the chromosome, whereas the lack of the functional UvrA or UvrB proteins causes substantial stabilization of (CTG/CAG) tracts.

scholarly journals Instability of Pathogenicity Islands in Uropathogenic Escherichia coli 536

2004 ◽  
Vol 186 (10) ◽  
pp. 3086-3096 ◽  
Author(s):  
Barbara Middendorf ◽  
Bianca Hochhut ◽  
Kristina Leipold ◽  
Ulrich Dobrindt ◽  
Gabriele Blum-Oehler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Direct Repeat ◽  
Pathogenicity Islands ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Derivatives Of ◽  
Specific Sequences

ABSTRACT The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.

scholarly journals Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

2017 ◽  
Author(s):  
María F Azpiroz ◽  
Magela D Laviña
Keyword(s):  
Escherichia Coli ◽  
Genomic Island ◽  
Direct Repeat ◽  
Host Cells ◽  
Dna Repeats ◽  
Direct Repeats ◽  
Site Specific ◽  
E Coli ◽  
A Site ◽  
New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

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