Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

1998 ◽  
Vol 260 (2-3) ◽  
pp. 226-231 ◽  
Author(s):  
S.-y. Xu ◽  
J.-p. Xiao ◽  
L. Ettwiller ◽  
M. Holden ◽  
J. Aliotta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cloning And Expression ◽  
Restriction Modification ◽  
Restriction Modification Systems

scholarly journals Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenic Escherichia coli

2021 ◽  
Author(s):  
Kurosh S Mehershahi ◽  
Swaine Chen
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Methylation ◽  
Gene Regulation ◽  
Detectable Effect ◽  
Epigenetic Mark ◽  
Type I ◽  
E Coli ◽  
Restriction Modification ◽  
Restriction Modification Systems

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life, extending also to bacterial virulence. Both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. However, the potential regulatory role of DNA methylation mediated by archetypal Type I systems in Escherichia coli has never been studied. We demonstrated that removal of DNA methylated mediated by three different Escherichia coli Type I RMSs in three distinct E. coli strains had no detectable effect on gene expression or growth in a screen of 1190 conditions. Additionally, deletion of the Type I RMS EcoUTI in UTI89, a prototypical cystitis strain of E. coli , which led to loss of methylation at >750 sites across the genome, had no detectable effect on virulence in a murine model of ascending urinary tract infection (UTI). Finally, introduction of two heterologous Type I RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of “regulation avoidance” for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome. Our results provide a clear and (currently) rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli , further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer.

DNA cytosine methyltransferase enhances viability during prolonged stationary phase in Escherichia coli

2020 ◽  
Vol 367 (20) ◽  
Author(s):  
Kevin T Militello ◽  
Lara Finnerty-Haggerty ◽  
Ooha Kambhampati ◽  
Rebecca Huss ◽  
Rachel Knapp
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Wild Type ◽  
Bacterial Physiology ◽  
Phase Competition ◽  
Cytosine Methyltransferase ◽  
The Impact ◽  
Restriction Modification ◽  
Restriction Modification Systems

ABSTRACT In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5′CCWGG3′ generating 5-methylcytosine. Dcm is not associated with a cognate restriction enzyme, suggesting Dcm impacts facets of bacterial physiology outside of restriction-modification systems. Other than gene expression changes, there are few phenotypes that have been identified in strains with natural or engineered Dcm loss, and thus Dcm function has remained an enigma. Herein, we demonstrate that Dcm does not impact bacterial growth under optimal and selected stress conditions. However, Dcm does impact viability in long-term stationary phase competition experiments. Dcm+ cells outcompete cells lacking dcm under different conditions. Dcm knockout cells have more RpoS-dependent HPII catalase activity than wild-type cells. Thus, the impact of Dcm on stationary phase may involve changes in RpoS activity. Overall, our data reveal a new role for Dcm during long-term stationary phase.

scholarly journals Transformation of Escherichia coli with the pGLO Plasmid: Going beyond the Kit

2019 ◽  
Vol 81 (1) ◽  
pp. 52-55 ◽  
Author(s):  
Charles E. Deutch
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Transformation Process ◽  
Host Restriction ◽  
Green Fluorescent ◽  
Restriction Modification ◽  
Restriction Modification Systems

The Bio-Rad pGLO bacterial transformation kit is commonly used to demonstrate this form of genetic exchange, which occurs in bacteria and eukaryotes and which differs fundamentally from transduction and conjugation. The basic experiment leads to the formation of green fluorescent colonies of Escherichia coli and can be extended to illustrate the specificity of the interaction between sugars and the AraC protein, the phenomenon of carbon catabolite repression, the substrate specificity of the β-lactamase encoded by the plasmid, and the role of host restriction/modification systems in the transformation process. pGLO DNA also can be isolated using plasmid mini-prep kits, analyzed with restriction endonucleases, and used to study the conditions for transformation in more detail.

scholarly journals Molecular Evolution of the Escherichia coli Chromosome. V. Recombination Patterns Among Strains of Diverse Origin

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 539-554 ◽  
Author(s):  
Roger Milkman ◽  
Elisabeth A Raleigh ◽  
Melissa McKane ◽  
Diane Cryderman ◽  
Patricia Bilodeau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Evolution ◽  
Parental Strain ◽  
Activity Analysis ◽  
Exonuclease Activity ◽  
Recombinant Chromosome ◽  
E Coli ◽  
Diverse Origin ◽  
Restriction Modification ◽  
Restriction Modification Systems

Abstract Incorporation patterns of donor DNA into recipient chromosomes following transduction or conjugation have been studied in the progeny of a variety of Escherichia coli crosses in which donor and recipient nucleotide sequences differ by 1-3%. Series of contiguous or variously spaced PCR fragments have been amplified from each recombinant chromosome and digested with a commercial restriction endonuclease previously shown to distinguish the respective parents in a given fragment. We conclude that entering donor DNA fragments are frequently abridged (cut and shortened) before incorporation, the cutting being due to restriction systems, and the shortening presumably due to exonuclease activity. Analysis of several backcrosses confirms, and extends to conjugation, the importance of restriction in E. coli recombination in nature. The transmission patterns in conjugation are similar to those of transduction, but (as expected) on a much larger scale. Asymmetric results of reciprocal crosses imply that mismatch frequency is not a major factor. Marked differences among the results of simple crosses according to parental strain combinations are consistent with observations that E. coli strains in nature vary dramatically in their restriction-modification systems.

scholarly journals Transduction, restriction and recombination patterns in Escherichia coli.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 35-43
Author(s):  
M McKane ◽  
R Milkman
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Chromosomal Dna ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Sequence Patterns ◽  
Single Marker ◽  
Different Strains ◽  
Restriction Modification ◽  
Restriction Modification Systems

Abstract Chromosomal DNA from several Escherichia coli reference (ECOR) strains was transduced by bacteriophage P1 into E. coli strain K12 W3110 trpA33. Recombination patterns of the transductants were determined by restriction fragment length polymorphism over a 40-kb region centering on a single marker (trpA+) in the tryptophan operon. These experiments demonstrate that transduction between different strains of E. coli can result in recombinational replacements that are small in comparison to the entrant molecule (replacements average 8-14 kb, whereas P1 packages approximately 100 kb) often in a series of discrete segments. The transduction patterns generated resemble the natural mosaic sequence patterns of the ECOR strains described in previous work. Extensive polymorphisms in the restriction-modification systems of the ECOR strains are a possible explanation for the sequence patterns in nature. To test this possibility two transductants were back-transduced into strain K12 W3110 trpA33. The resulting patterns were strikingly different from the original transductions. The size of the replacements was greater, and no multiple replacements were observed, suggesting a role for restriction-modification systems in the transduction patterns and perhaps for the mosaic sequence patterns in nature.

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3331-3341 ◽  
Author(s):  
Iwona Mruk ◽  
Magdalena Cichowicz ◽  
Tadeusz Kaczorowski
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Start Codon ◽  
Sequence Motifs ◽  
Amino Acid Sequence Level ◽  
Specific Sequence ◽  
Strong Inhibitor ◽  
Apparent Homogeneity ◽  
Restriction Modification ◽  
Restriction Modification Systems

The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M r 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 5′-AAGCTT-3′. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 5′ end of the specific sequence to N 6-methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction–modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N 6-adenine β-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg2+ for phosphodiester bond cleavage. Mg2+ was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg2+ may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed.

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