Dcm Mutants Research Articles

Improving transformation of Staphylococcus aureus belonging to the CC1, CC5 and CC8 clonal complexes.

Methicillin resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen found in hospital and community environments that can cause serious infections. A major barrier to genetic manipulations of clinical isolates has been the considerable difficulty in transforming these strains with foreign plasmids, such as those from E. coli, in part due to the type I and IV Restriction Modification (R-M) barriers. Here we combine a Plasmid Artificial Modification (PAM) system with DC10B E. coli cells (dcm mutants) to bypass the barriers of both type I and IV R-M of S. aureus, thus allowing E. coli plasmid DNA to be transformed directly into clinical MRSA strains MW2, N315 and LAC, representing three of the most common clonal complexes. Successful transformation of clinical S. aureus isolates with E. coli-derived plasmids should greatly increase the ability to genetically modify relevant S. aureus strains and advance our understanding of S. aureus pathogenesis.

DCM Mutation ACTCE361G Causes Uncoupling of Myofibril Sensitivity from TnI Phosphorylation that can be Reversed by Epigallocatechin-3-Gallate

We examined the relationships between troponin I phosphorylation and Ca2+-regulation of contractility in single myofibrils from a mouse model of familial DCM (ACTC E361G) in comparison with non-transgenic (NTG). We measured the effects of changing [Ca2+] and troponin I phosphorylation level on myofibril contractility. Propranolol treatment of mice was used to reduce the level of troponin I in their hearts prior to isolating the myofibrils.For non-transgenic mouse myofibrils we found that when the TnI phosphorylation level was reduced from 1.02 to 0.3 the Ca2+-sensitivity of force was increased (EC50P/unP=1.8), relaxation parameter kREL was reduced and tLIN was increased. ACTCE361G mouse myofibrils were uncoupled: Ca2+-sensitivity and relaxation parameters did not depend on troponin I phosphorylation level (EC50P/unP=0.88). Nevertheless, modulation of Ca2+-sensitivity by sarcomere length or due to EMD57033 was retained.The Ca2+-desensitiser Epigallocatechin-3-gallate (EGCG) decreased Ca2+-sensitivity in phosphorylated and unphosphorylated NTG myofibrils equally (EC50 P/uP= 0.50±0.06 and 0.45±0.07 respectively) but did not change the relaxation parameters tLIN and kRE. The rate of force development (kACT), measured at high Ca2+, was unchanged in myofibrils with phosphorylated TnI and 22% decreased in unphosphorylated myofibrils indicating that EGCG affects cross-bridge activation kinetics.EGCG reduced Ca2+-sensitivity and kACT in both phosphorylated and unphosphorylated ACTCE361G myofibrils. The change in EC50 was more in phosphorylated than unphosphorylated myofibrils, consequently EGCG restored the lost difference in EC50 values between phosphorylated and unphosphorylated myofibrils and also the difference in relaxation parameters tLIN and kREL. The observation that EGCG does not affect either the EC50 P/ EC50 unP or tLIN in NTG myofibrils but changes them in ACTG E361G suggests that EGCG can restore modulation of cardiac contractile function by TnI phosphorylation to DCM mutant myofibrils independently of its Ca2+-desensitising function.

Biochemical characterisation of Troponin C mutations causing hypertrophic and dilated cardiomyopathies.

Cardiac muscle contraction occurs through an interaction of the myosin head with the actin filaments, a process which is regulated by the troponin complex together with tropomyosin and is Ca(2+) dependent. Mutations in genes encoding sarcomeric proteins are a common cause of familial hypertrophic and dilated cardiomyopathies. The scope of this review is to gather information from studies regarding the in vitro characterisation of six HCM and six DCM mutations on the cardiac TnC gene and to suggest, if possible, how they may lead to dysfunction. Since TnC is the subunit responsible for Ca(2+) binding, mutations in the TnC could possibly have a strong impact on Ca(2+) binding affinities. Furthermore, the interactions of mutant TnCs with their binding partners could be altered. From the characterisation studies available to date, we can conclude that the HCM mutations on TnC increase significantly the Ca(2+) sensitivity of force development or of ATPase activity, producing large pCa shifts in comparison to WT TnC. In contrast, the DCM mutations on TnC have a tendency to decrease the Ca(2+) sensitivity of force development or of ATPase activity in comparison to WT TnC. Furthermore, the DCM mutants of TnC are not responsive to the TnI phosphorylation signal resulting in filaments that preserve their Ca(2+) sensitivity in contrast to WT filaments that experience a decrease in Ca(2+) sensitivity upon TnI phosphorylation.

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca2+, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T HC (high Ca2+ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T a, ±SEM, N = 34; E54K: 1.24±0.07 T a, N = 28), a significantly lower T LC (low- Ca2+ tension at pCa 7.0) (E40K: 0.07±0.02 T a, N = 34; E54K: 0.06±0.02 T a, N = 28), and a significantly lower T act (Ca2+ activatable tension) (T act = T HC–TLC, E40K: 1.15±0.08 T a, N = 34; E54K: 1.18±0.06 T a, N = 28) than WT (T HC = 1.53±0.07 T a, T LC = 0.12±0.01 T a, T act = 1.40±0.07 T a, N = 25). All tensions were normalized to T a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca2+ sensitivity (pCa50) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.

Functional genomics of dichloromethane utilization in Methylobacterium extorquens DM4

Dichloromethane (CH(2)Cl(2) , DCM) is a chlorinated solvent mainly produced by industry, and a common pollutant. Some aerobic methylotrophic bacteria are able to grow with this chlorinated methane as their sole carbon and energy source, using a DCM dehalogenase/glutathione S-transferase encoded by dcmA to transform DCM into two molecules of HCl and one molecule of formaldehyde, a toxic intermediate of methylotrophic metabolism. In Methylobacterium extorquens DM4 of known genome sequence, dcmA lies on a 126 kb dcm genomic island not found so far in other DCM-dechlorinating strains. An experimental search for the molecular determinants involved in specific cellular responses of strain DM4 growing with DCM was performed. Random mutagenesis with a minitransposon containing a promoterless reporter gfp gene yielded 25 dcm mutants with a specific DCM-associated phenotype. Differential proteomic analysis of cultures grown with DCM and with methanol defined 38 differentially abundant proteins. The 5.5 kb dcm islet directly involved in DCM dehalogenation is the only one of seven gene clusters specific to the DCM response to be localized within the dcm genomic island. The DCM response was shown to involve mainly the core genome of Methylobacterium extorquens, providing new insights on DCM-dependent adjustments of C1 metabolism and gene regulation, and suggesting a specific stress response of Methylobacterium during growth with DCM. Fatty acid, hopanoid and peptidoglycan metabolisms were affected, hinting at the membrane-active effects of DCM due to its solvent properties. A chloride-induced efflux transporter termed CliABC was also newly identified. Thus, DCM dechlorination driven by the dcm islet elicits a complex adaptive response encoded by the core genome common to dechlorinating as well as non-dechlorinating Methylobacterium strains.

The Role of Cardiac Troponin T Quantity and Function in Cardiac Development and Dilated Cardiomyopathy

BackgroundHypertrophic (HCM) and dilated (DCM) cardiomyopathies result from sarcomeric protein mutations, including cardiac troponin T (cTnT, TNNT2). We determined whether TNNT2 mutations cause cardiomyopathies by altering cTnT function or quantity; whether the severity of DCM is related to the ratio of mutant to wildtype cTnT; whether Ca2+ desensitization occurs in DCM; and whether absence of cTnT impairs early embryonic cardiogenesis.Methods and FindingsWe ablated Tnnt2 to produce heterozygous Tnnt2 +/− mice, and crossbreeding produced homozygous null Tnnt2 −/− embryos. We also generated transgenic mice overexpressing wildtype (TGWT) or DCM mutant (TGK210Δ) Tnnt2. Crossbreeding produced mice lacking one allele of Tnnt2, but carrying wildtype (Tnnt2 +/−/TGWT) or mutant (Tnnt2 +/−/TGK210Δ) transgenes. Tnnt2 +/− mice relative to wildtype had significantly reduced transcript (0.82±0.06[SD] vs. 1.00±0.12 arbitrary units; p = 0.025), but not protein (1.01±0.20 vs. 1.00±0.13 arbitrary units; p = 0.44). Tnnt2 +/− mice had normal hearts (histology, mass, left ventricular end diastolic diameter [LVEDD], fractional shortening [FS]). Moreover, whereas Tnnt2 +/−/TGK210Δ mice had severe DCM, TGK210Δ mice had only mild DCM (FS 18±4 vs. 29±7%; p<0.01). The difference in severity of DCM may be attributable to a greater ratio of mutant to wildtype Tnnt2 transcript in Tnnt2 +/−/TGK210Δ relative to TGK210Δ mice (2.42±0.08, p = 0.03). Tnnt2 +/−/TGK210Δ muscle showed Ca2+ desensitization (pCa50 = 5.34±0.08 vs. 5.58±0.03 at sarcomere length 1.9 µm, p<0.01), but no difference in maximum force generation. Day 9.5 Tnnt2 −/− embryos had normally looped hearts, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres.ConclusionsAbsence of one Tnnt2 allele leads to a mild deficit in transcript but not protein, leading to a normal cardiac phenotype. DCM results from abnormal function of a mutant protein, which is associated with myocyte Ca2+ desensitization. The severity of DCM depends on the ratio of mutant to wildtype Tnnt2 transcript. cTnT is essential for sarcomere formation, but normal embryonic heart looping occurs without contractile activity.

Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse α-cardiac myosin in the laser trap assay

Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity (F(max)) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. F(max) was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas F(max) was severely depressed for one of the DCM mutants (65% S532P). Although F(max) was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity (V(actin)) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These F(max) data combined with previous V(actin) measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM.

DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

Efficient transformation of Mesorhizobium huakuii subsp. rengei and Rhizobium species

The transformation of Mesorhizobium huakuii subsp. rengei B3 with either pBBR122 or pKT230 was carried out. We determined the optimal conditions required for transformation by electroporation and obtained up to 10 5 CFU/μg pBBR122. Plasmids prepared from strain B3 yielded higher transformation efficiency than those from various dam or dcm mutant strains of Escherichia coli. This result suggests that a high transformation efficiency is not related to the methylation of plasmid DNA in E. coli. Using the optimal conditions for electroporation, we performed transformation of several species of Rhizobium, Mesorhizobium and Sinorhizobium. All tested strains of these species were transformed with pBBR122. Strains of M. huakuii and R. phaseoli AHU1133 were transformed at high efficiency, whereas transformation efficiencies of Rhizobium sp. NGR234 and S. meliloti strains were less than 2 × 10 3 CFU/ μg plasmid DNA.

Very short patch mismatch repair activity associated with gene dcm is not conferred by a plasmid coding for EcoRII methylase.

The only cytosine methylase in Escherichia coli K-12 methylates the second cytosine in the sequence CC (A/T)GG and is encoded by gene dcm. Methylation and very short patch mismatch repair activities lacking in a dcm mutant of E. coli were restored by a plasmid containing the cloned dcm gene. In contrast, plasmids with the gene for EcoRII methylase, which is a homolog of dcm, restored only cytosine methylase activity and not mismatch repair.

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of λ prophage in Dcm + strains but not in Dcm − mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm + or Dcm − strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA : V. Recombinogenic plasmids from arl mutants of Escherichia coli are unusually sensitive to nuclease S 1 and partially deficient in cytosine methylation at C-C-(A/T)-G-G sequences

Two novel phenotypes previously associated with arl mutations of Escherichia coli, increased frequencies of genetic recombination and unusual sensitivity of DNA to the single-strand-specific nuclease S 1, have been defined most completely by the properties of λ bacteriophages grown on arl bacteria (Arl − phages). We now find that plasmids maintained in arl mutants (Arl − plasmids) exhibit elevated recombination frequencies, unusual sensitivity to nuclease S 1 (in a limited number of regions) and a new Arl phenotype, partially deficient methylation of the inner cytosine at C-C-(A/T)-G-G sequences. A variety of Arl − plasmids (all pBR322 derivatives) show elevated recombination (4 to 10-fold) by three different assays (frequencies of homomultimers and of heteromultimers, efficiency of intramolecular recombination). Plasmids from arl bacteria (after conversion to linear form) are nicked by nuclease S 1 about 0.7 times per duplex; Arl + plasmids are nuclease S 1-resistant. Restriction endonuclease EcoRII (recognition sequence, C-C-(A/T)-G-G) cuts Arl − plasmid DNA more readily than Arl + DNA, but Arl − plasmids are still more EcoRII-resistant than Dcm − plasmids (from E. coli dcm mutants, which lack the chromosomal cytosine methylase; recognition sequence, also C-C-(A/T)-G-G). By chromatographic analyses, Arl − plasmid DNA contains less 5-methylcytosine than Arl + (0.07% versus 0.15%). although the 6-methyladenine content is the same (0.5mol%).

Partially deficient methylation of cytosine in DNA at CC TAGG sites stimulates genetic recombination of bacteriophage lambda

Lambda bacteriophages grown on arl mutants of Escherichia coil (“Arl −” phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m 5C) than phages grown on wild-type bacteria (“Arl +” phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm 5C T AGG) characteristic of E. coil K-12 bacteria (“Dcm −” phages). “Arl −” phages are one twelfth as resistant to Eco RII restriction (recognition site CC T AGG) as “Arl +” phages, but 40-fold more resistant than “Dcm −” phages. Chromatographic analyses show the 5-methylcytosine content of “Arl −” DNA to be one third that of “Arl +” DNA. Altered cytosine methylation frequency correlates with two previously described properties of “Arl −” phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria. Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from “Dcm −” phages) is highly recombinogenic but not S1-sensitive. We hypothesize that hemimethylated CC T AGG sites in “Arl −” DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.

Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12.

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.