DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward-or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context-and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.
The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grow on dihydrogen-fumarate medium and also produced very low levels of HIYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3.The hyb operon was mapped at 65 min on the E. coli chromosome.Under anaerobic growth conditions, Escherichia coli produces three different nickel-containing hydrogenases (3, 39). Hydrogenase 3 (HYD3) is part of the formate hydrogenlyase (FHL) complex and is responsible for formate-dependent dihydrogen (H2) evolution. The operon encoding HYD3 and other accessory electron transport components of the FHL complex, hyc, has been identified and is located at 58 min on the E. coli chromosome (7). The highly oxygen-labile nature of HYD3 has precluded detailed biochemical characterization. HYD2 is involved in H2 uptake and can be differentially induced to high levels when cells are grown in medium containing H2 as an electron donor and fumarate as an electron acceptor (3,23,25,39). An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4). Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1, 16). The operon encoding the two structural subunits of HYD1 (hya) contains a total of six genes and has been mapped at 22 min on the E. coli chromosome (30,31). The function of HYD1 is not understood, but it is believed to have a role in hydrogen cycling during fermentative growth. In addition to the operons coding for the structural components of the three hydrogenases, a fourth operon, hyp, located at 58 min, is essential for activity of all three hydrogenases (20,26,38). At least one of the genes in this operon (hypB) is involved in nickel metabolism, most probably via nickel insertion into apoenzyme (27).In this paper, we present the DNA sequence of the operon encoding HYD2 (hyb), which contains seven open reading frames (ORFs). Cassette mutagenesis of the hyb operon on the chromosome resulted in a total loss of HYD2 expression and activity, as well as in significant reduction in HYD1 activity. MATERIALS AND METHODSBacterial strains. All bacterial strains used were E. coli K-12 derivatives and are listed in Table 1 [pH 7.0]), resuspended in the same buffer to an optical density of 0.5 at 600 nm, and used for whole-cell enzyme assays. Cell extracts were prepared by sonicating cell suspensions on ice with a model W385 sonicator (Heat Systems) for 20 5-s bursts. Triton X-100 was added to a final concentration of 2% (vol/vol), when req...
Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein.The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYDl activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.Hydrogen metabolism in Escherichia coli is tightly regulated by parameters of growth (2, 27) and involves three discrete nickel-containing hydrogenases: two electrophoretically stable, membrane-bound heterodimeric enzymes, hydrogenase 1 (HYD1) (28) and hydrogenase 2 (HYD2) (3), and a labile hydrogen-evolving hydrogenase, hydrogenase 3, which is as yet uncharacterized (27). HYD1 has been purified from anaerobically grown cells and biochemically characterized (28). It has a molecular mass of 200 kDa, is composed of two large (60-kDa) and two small (32-kDa) subunits, and contains 11 nonheme iron atoms and 1 g-atom of nickel per mol of enzyme (28 (16,24,25,30,33); and (iii) mutants unable to utilize hydrogen in the presence of electron acceptors (16,32). None of the mutants, however, have been shown to specifically impair HYD1 activity, indicating that the mutations are not in the hya operon. In this paper, we report the construction, biochemical characterization, and genetic analysis of HYD1-specific deletion mutants. MATERIALS AND METHODSBacterial strains and culture conditions. All bacterial strains used in this study are derivatives of E. coli K-12 and are listed in Table 1. Bacteria were cultured in Luria broth with 0.4% glucose as the carbon source (LBG). Antibiotics were added at final concentrations of 50 jig/ml (kanamycin), 100 jig/ml (ampicillin), and 20 ,ug/ml (chloramphenicol). For studying the incorporation of nickel into HYD1, cells were grown anaerobically in LBG in the presence of 1.2 ,iM...
We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.
A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system.
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